Up-regulation of heparin binding epidermal growth factor-like growth factor and amphiregulin...

ALIMENTARY TRACT DIGEST LIVER IJIS 2002~3~:~SS-505

Up-regulation of heparin binding epidermal growth factor-like growth factor and amphiregulin expression in Helicobacter pylori-infected human gastric mucosa

C. Tuccillol 4* B.A. Manzol* G. Nardone2 G. D’Argenio3 A. Rocco2 A. Di Popolo4 N. Della Valle3 S. Staibano5 G. De Rosa5 V. RiccF C. Del Vecchio Blancol R. Zarrilli4 M. Romanol 4

From I Department of Medicine, Gasfroenterology, Second University of Naples, Naples; 2 Hepatology Unit, 3 Gastroenterology Unit. 4 L?epartment of Cellular and Molecular Biology and Pathology ‘L. Celifano’Y 5 Department of Biomorphologic and Functional Sciences, Pathology Section, “Federico II” University, Naples; 6 institute of Human Physiology, University of Pavia, Italy; * C. T. and B.A. M. contributed equally fo this work.

af/6hws fsf comw~&lmt? Dr. M. Romeno, Dipartimento di lnternistica Clinica e Sperimentale - Gastroenterologia, Seconda Universitd di Napoli, c/o II Policlinico, Padiglione 3, Second0 Piano, via Pansini 5, 80131 Napoli, I&iy. Fax: +39-081-5666714. E-mail: marco. romano@unine2. it

achnswl$dgemmlb Work supported in part by grants from Comitato Nazionale Bioeecnologie e Bologie Molecolara of CNR, M/L/t?, CIRANALJ of II Universita di Napoli, and 5YK GULUEN, Italy, Authors thank M. Berardone and 8. Teocle for artwork and Dr. RX. Harris, Vanderbilt University Medical School, Nashville, TN, USA, for providing antibody to human HE-EGF,

Submitted December 31. 2001. Accepted after revision, February 22, 2002.

Background. Host response plays a major role in pathogenesis of He- licobacter pylori-induced gastroduodenal disease including adenocarcinoma of distal stomach. Epidermal growth factor-related growth factors are important modulators of gastric homeostasis in normal and damaged gastrointestinal mucosa. Aim. To evaluate expression of heparin binding epidermal growth factor and amphiregulin in antral mucosa of Helicobacter pylori-infected and non-infected dyspeptic patients and to correlate levels of heparin binding-epidermal growth factor and amphiregulin mRNA with mito- genie activity of gastric epithelial cells. Methods. A total of IO Helicobacter pylori-infected and 15 Helicobac- ter pylori non-infected [IO with and 5 without gastritis] dyspeptic patients were studied. Diagnosis of Helicobacter pylori infection was based on rapid urease test and histology Heparin binding-epidermal growth factor and amphiregulin mRNA expression in antral mucosa were assessed by reverse transcriptase-polymerase chain reaction. Protein expression and localization of both peptides were determined by immunohistochemistry. Mitogenic activity of antral gastric mucosa was assessed by determination of proliferating cell nuclear antigen labelling index by immunohistochemistry. Results. Heparin binding-epidermal growth factor and amphiregulin mRNA expression increased in Helicobacter pylori-infected vs Heli- cobacter pylori non-infected patients. Heparin binding-epidermal growth factor and amphiregulin immunostaining was more intense and deeper in gastric gland compartment in infected mucosa than in non- infected mucosa. Increase in heparin binding-epidermal growth factor and amphiregulin mRNA expression significantly correlated with increase in proliferating cell nuclear antigen labelling index. Conclusions. Helicobacter pylori gastritis is associated with up-regulation of heparin binding-epidermal growth factor and amphiregulin which correlates with increased mitogenic activity of gastric mucosa. In- creased heparin binding-epidermal growth factor and amphiregulin expression is postulated to contribute to reparative response of gastric mucosa to Helicobacter pylori infection.

Digest Liver Dis 2002;34:499-505

Key words: amphiregulin; gastric mucosa; Helicobacter py/ori; heparin binding-epidermal growth factor; proliferating cell nuclear antigen

Introduction

Helicobacter pylori (H. pylori) is the major causative agent of chronic superficial gastritis and peptic ulcer disease and seems to play an important role in the development of adenocarcinoma of the distal stomach in humans lm3. The mechanisms whereby H. pylori may cause gastroduodenal

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disease and contribute to gastric carcinogenesis are still hypothetical 4. However, production by the bacterium of specific virulence factors that cause damage to the epithelial cells and impair the healing process of the damaged mucosa together with the response of the host to the infection may be involved4. Polypeptide growth factors, and, in particular, epider- ma1 growth factor (EGF)-related growth factors, are important modulators of gastric homeostasis in normal and damaged gastrointestinal (GI) mucosa and play a crucial role in the reparative events following gastric mucosal injury . 5-8 Moreover, altered expression of EGF-related peptides has been reported in GI malignancies in humans . 9-‘2 There are still controver- sial aspects about the possible relationship between H. pylori infection and EGF-related growth factors. We have recently shown that the expression of heparin binding EGF-like growth factor (HB-EGF) and amphiregulin (AR), members of the EGF receptor family of ligands, is up-regulated in human gastric epithelial cells exposed to H. pylori, whereas transforming growth factor CI (TGF-a) is not 13. Moreover, we have previously shown that the tissue concentration of TGF-a is decreased in H. pylori-infected human gastric mucosa ‘I. On the other hand, Schiemann et al. have recently shown a decrease in HB-EGF, AR, and TGF-a mRNA expression in H. pylori-infected human gastric mucosa Is. This study was, therefore, designed to evaluate the expression of HB-EGF and AR in the human gastric mucosa from H. pylori-infected and non-infected dyspeptic patients and to correlate the levels of HB-EGF and AR mRNA with the mitogenic activity of gastric epithelial cells as assessed by determination of proliferating cell nuclear antigen (PCNA), a well-established marker of cell proliferation 16.

Patients and methods

Patients We studied 25 patients referred to our endoscopy units on account of dyspepsia. Ten (6 male and 4 female, age range 42-63 years, median 46) were H. pylori-infected and 15 were H. pylori non-infected. Of the 15 H. pylori-non-infected subjects, 10 (5 male and 5 female, age range 31-54 years, median 38) had some degree of gross gastric injury at endoscopy and chronic gastritis at histology, while 5 (3 male and 2 female, age range 25-42 years, median 31) had completely normal stomach both at endoscopy and histology. Diagnosis of H. pylori infection was based on rapid urease test and histology. Two biopsy samples were taken from the gastric antrum, body and angulus for histology. Haematoxylin and eosin staining was used for histological assessment

and a modified Giemsa staining was used for H. pylori identification. For determination of HB-EGF and AR mRNA expression, 4 biopsy samples were taken from the antrum, snap frozen in liquid nitrogen and stored at -70°C until assayed. A blood sample was obtained from each of the H. pylori-infected subjects to deter- mine the CagA status by Western blotting (Helico 2.0, Genelabs Diagnostics, Singapore). None of the subjects had been drinking alcohol or taking non-steroidal anti- inflammatory or anti-secretory drugs for at least 15 days prior to endoscopy. Each patient gave his/her in- formed consent to enter the study. The procedures followed in this study were in accordance with the ethical standards of the committee on human experimentation of the institution in which they were done and in accordance with the Declaration of Helsinki.

Reverse transcriptase-polymerase chain reaction (RT- PCR) analysis RT-PCR analysis was performed on total RNA isolated from gastric antral biopsies as described previously I7 lx. First-strand complementary DNA was prepared using 200 units of reverse transcriptase (Supertranscript RT, Gibco BRL, Gaithersburg, MD, USA), 1 ug of total RNA as template, and 10 pmol/l of random hexamers in the presence of 0.1 mmol/l dithiothreitol, 0.5 mmol/l dNTP-litium salt (Pharmacia, Milan, Italy), and 20 units of RNase inhibitor (Promega, Madison, WI, USA). The reaction profile was 37°C x 10 minutes, followed by 42°C x 60 minutes. To control for contamination by genomic DNA, all RNA samples were run in duplicate with or without addition of reverse transcriptase. Semiquantitative RT-PCR coamplification of HB- EGF/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and AR/GAPDH transcripts was performed using HB-EGF (sense, GCCACACCAAACAAG- GAGGAGCAC; antisense, AGATGACAGCACAGC- CACCAC), AR (sense, CCTTCTAGTAGTGAAC- CGTCCTCG; antisense, GCATGTTACT- GCTTCCAGGTGCTC), and GAPDH (sense, CAC- CATCTTCCAGGAGCGAG; antisense, TCACGC- CACAGTTTCCCGGA)-specific primers. Primers were placed on different exons. Primers were designed on the basis of the coding sequences of the human HB- EGF or AR mRNA (accession number M60278 and M30704, respectively). GAPDH primers were as previously described I7 ‘*. All primers were provided by PRIMM Srl (Milan, Italy) and were used at the final concentration of 2 mmol/l. PCR amplifications were performed using 50 ng of cDNA in the presence of 0.2 mM dNTP (Boehringer Mannheim, Germany), 1.5 yCi o- 32P dGTP, and 0.3 ul AmpliTaq DNA polymerase (Perkin Elmer, Branchburg, NJ, USA). MgC12 was added at the final concentration of 1.5 mmol/l. After an

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HG-fGF and AR expression in H. pylorigastritis

initial denaturation step, 95°C x 2 minutes, the PCR amplification was performed using the following pro- files: HB-EGF/GAPDH: 94°C x 1 minute, 62°C x 1 minute, and 72°C x 1 minute for 30 cycles. GAPDH primers were added after 7 cycles. AIUGAPDH: 94°C x 1 minute, 54°C x 1 minute, and 72°C x 1 minute for 28 cycles. GAPDH primers were added after 5 cycles. In all cases, the final extension was at 72°C x 10 minutes. PCR products were phenol-chloroform extracted, ethanol precipitated and subjected to 6 and 4% poly- acrylamide gel electrophoresis, respectively, and au- toradiography. Sizes of the amplified fragments were estimated from migration of the 1 -kb ladder molecular- weight marker (Gibco Laboratories, Grand Island, NY, USA), and identity was assessed by restriction-enzyme digestion. To test for contamination by genomic DNA, samples were run in duplicate with (+RT) or without (-RT) the addition of reverse transcriptase. PCR products were quantified by densitometric scanning of the autoradiograms using a Howteck Scanmaster 3 densitometric with RFL Print-TM Software (Pharmacia, Biotech Inc, Cologno Monzese, Italy). HB-EGF and AR mRNA levels were expressed as arbitrary units of optical density. Each individual value of mRNA levels represents the mean value of three independent RT- PCRs. HB-EGF and AR transcripts levels were normalised to those of GAPDH.

HB-EGF and AR immmunolocalisation Immunohistochemistry was performed as previously described i9. In brief, 4u thick serial sections were cut from paraffin blocks, mounted on acid-cleaned glass slides, and heated at 55°C for 60 min. Slides were de- waxed and rehydrated, then the endogenous peroxidase activity was inhibited by incubation with 0.3% Hz02 in methanol (20 min at room temperature). To re- duce non-specific background staining, slides were incubated with 5% horse serum (15 min at room temperature). To enhance immunostaining, sections were treated with an antigen retrieval solution (10 mM citric acid monohydrate, pH 6.0, adjusted with 2N NaOH) and heated three times in a microwave oven at high power for live minutes. Rabbit anti-human HB-EGF polyclonal antibody 2998, kindly provided by Dr. R.C. Harris, Vanderbilt University, Nashville, TN, USA, and rabbit anti-human AR polyclonal antibody (Neo-Mark- ers, Union City, CA, USA) were used at 1: 1000 and 1:50 dilution in phosphate buffer solution (PBS), respectively. Incubation was performed for 1 hour at room temperature. Slides were then washed in PBS for 5 minutes and incubated with biotinylated anti-rabbit IgG antibody for 40 minutes at room temperature. Af- ter washing with PBS, sections were incubated with avidin-biotin complex (ABC standard, Vector Labora- tories Inc., Burlingame, CA, USA). Peroxidase conju-

gates were subsequently localised by diaminobenzi- dine tetrahydrochloride as a chromogen. Sections were counterstained with Harris’s haematoxylin. Antibody specificity was confirmed by preabsorption of anti- HB-EGF and anti-AR antibodies with recombinant human (rh) HB-EGF (Sigma Chemical Co., St Louis, MO, USA) or rhAR (R&D Systems, Inc., Minneapolis, MN, USA) and by incubation without the primary an- tiserum.

Mitogenic activity Gastric epithelial cell mitogenic activity was determined using an immunohistochemical staining for PC- NA as previously described 19. PCNA is a well-established marker of cell proliferation I6 I9 2o that, in our hands, correlates with other markers of gastric epithelial cell mitogenesis such as AgNOR 19. Tissue samples were processed as described above. Slides were then incubated in a moist chamber overnight at 4°C with monoclonal primary antibody anti-PCNA (anti-PCNA clone PClO; Upstate Biotechnology, Lake Placid, NJ, USA, dilution 1:500). Visualization of immunoreac- tion was as described above. Negative controls with human non-immune serum substituting for specific primary antibody were included in each run. A human reactive lymph node was the positive control for PC- NA. PCNA labelling index was determined by two independent observers (A.R. and S.S.) blinded to H. py- Zori status by counting PCNA positive cells in at least five random fields of 300 cells each and expressed as percent of PCNA immunoreactive cells. The interob- server agreement was excellent with a k coefficient value of 0.75.

Statistical analysis Significance of differences was evaluated by Wilcoxon rank sum test. Correlation was assessed by regression analysis. Significance was defined as a p value of ~0.05.

Results

Endoscopic and histological features Of the H. pylori non-infected patients, 8 had antral erythema, 2 had gastroduodenal erosions and 5 had no macroscopic injury at endoscopy. At histology, 9 had mild to moderate chronic non-active superficial gastritis, one showed mild chronic active gastritis, and 5 had normal histology. Of the 10 H. pylori infected patients, 4 had antral erythema, 5 had gastroduodenal erosions and 1 had duodenal ulcer at endoscopy. At histology, all the H. pylori infected subjects had mild to moderate chronic active superficial gastritis, with intestinal metaplasia in one, and mild to moderate H. pylori density. Seven H. pylori-infect-

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ed subjects showed positivity for anti-CagA antibody while 3 did not.

HB-EGF and AR mRNA expression HB-EGF and AR mRNA expression, as assessed by semiquantitative RT-PCR coamplification of HB-

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Fig. 1. HB-EGF and AR mRNA expression in human antral gastritis. a: Semiquantitative RT-PCR coamplification of HB-EGF/GAPDH and AWGAPDH transcripts in non-infected [Hp-I or H. pylori-infected [HP+’ gastric biopsies. HB-EGF/GAPDH and AWGAPDH PCR products were separated by electmphorasis on 6% and 4% polyacry- lamide gals, respectively. To test for contamination by genomic DNA, samples were run in duplicate with f+RTI or without f-RTI the addition of reverse trenscriptase. Representative autoradiograms are shown. Amplified PCR products ate indicated on left side of autoradiograms. Sizes are shown on right. b: Densitometric scan values of HB-EGF and AR mRNA levels in gastric biopsies fmm H. pybri non- infected (Hp.1 or H. py/ori-infected fHp+I patients. HE-EGF and AR transcript levels are normalised to those of GAPDH and are expressed as arbitrary units Median densitometric scan values of each group are indicated by horizontal bars. Abbreviations: see list.

EGF/GAPDH and ARZGAPDH transcripts, was detectable in the antral gastric mucosa from non-infected dyspeptic subjects and was significantly higher in H. pylori-infected patients (Fig. 1, panel A). In particular, HB-EGF mRNA levels were 2.5-fold higher in H. py- Zori-infected compared with non-infected subjects with gastritis (median 1.15, range 0.62-1.45 HB- EGF/GAPDH arbitrary units vs median 0.46, range 0.20-0.70 HB-EGF/GAPDH arbitrary units, respectively, p<O.OOl) (Fig. 1, panel B). Also, AR mRNA expression was 2-fold higher in H. pylori infected than in non-infected subjects with gastritis (median 0.95, range 0.65- 1.30 ARZGAPDH arbitrary units vs median 0.47, range 0.16-0.67 ARZGAPDH arbitrary units, respectively, p<O.OOl) (Fig. 1, panel B). All subjects but one in the H. pylori-infected group had HB-EGF and AR mRNA levels higher than the highest value in the non-infected group. The lowest levels of mRNA expression for both growth factors in the H. pylori-infected group were observed in the only patient with duodenal ulcer. The 5 H. pylori non-infected subjects with endoscopically and histologically normal gastric mucosa had HB-EGF and AR mRNA levels compara- ble to those observed in H. pylori non-infected subjects with gastritis (data not shown). In particular, HB-EGF mRNA levels were: median 0.32, range 0.15-0.54 HB- EGF/GAPDH arbitrary units and AR mRNA levels were: median 0.34, range 0.1 l-0.50 AIUGAPDH arbitrary units.

Immunolocalisation of HB-EGF and AR We also studied expression and localization of immunoreactive HB-EGF and AR in the gastric antrum of subjects with and without H. pylori infection (Fig. 2). An increase in the intensity of the immunostaining for HB-EGF and AR was observed in H. pylori-infected gastric mucosa compared with non-infected mucosa (Fig. 2, A vs C and B vs D, respectively). Both peptides showed a cytoplasmic localization with a reinforce- ment of the signal at the cell membrane level (Fig. 2 C, D). Moreover, in H. pylori-infected subjects, the immunostaining for HB-EGF and AR was detectable not only in the superficial epithelium (as mainly observed in H. pylori non-infected subjects) but also deeper in the glandular compartment of the mucosa (Fig. 2 C, D). A number of cells showed nuclear immunoreactivity for AR in the glandular compartment of H. pylori- infected mucosa but not in the non-infected mucosa (Fig. 2, B vs D).

Mitogenic activity To assess the mitogenic activity of the gastric mucosa, we evaluated the number of PCNA positive cells by immunohistochemistry. PCNA labelling index was slightly but significantly higher in H. pylori-infected

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HE-ECF and AR expression in K py/wi gastritis

Fig. 2. Locaiisation of immunoreactive HB-EGF and AR in the gastric antrum of dyspeptic subjects with and without H. py/ori infection. 3 and 6. HB- EGF and AR immunostaining, respectively, in ff. py/ori negative chronic gastritis (original magn. 400x and 250x, respectively]. G and II. HB-EGF and AR immunostaining, respectively, in H. py/ori positive chronic gastritis, [original magn. 400x). Arrowheads indicate nuclear AR immunoreactivity. Ab- breviations: see list.

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Fig. 3. Mitogenic activity of gastric mucosa in H. pylori positive Wp+l and negative U-/p1 gastritis as assessed by determination of PCNA labelling index. Median value in each group is represented by horizontal bars. Abbreviations: see list.

gastric mucosa than it was in non-infected mucosa with gastritis (median 30.6%, range 27.5%-32.5% vs median 27.8%, range 26.6%-30.1%, ~~0.05) (Fig. 3). PCNA labelling index in the 5 H. pylori negative subjects with completely normal stomach was lower than that observed in H. pylori negative subjects with gastritis (i.e., median 26.4%, range 24.1%-29.4%), but this did not reach statistical significance. The PCNA labelling index significantly correlated with HB-EGF or AR mRNA expression as assessed by linear regression analysis. (r=0.93, p<O.OOl and r=0.94, p<O.OOl, respectively)

Discussion

This study shows an increased expression of HB-EGF and AR in H. pylori-infected vs H. pylori non-infected

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patients. The increase in HB-EGF and AR mRNA expression significantly correlated with the increase in the PCNA labelling index. H. pylori infection causes gastritis and gastroduodenal mucosal ulceration and is associated with an increased risk for development of adenocarcinoma of the distal stomach in humans ‘-3. Specific virulence factors pro- duced by the bacterium contribute to the development of gastroduodenal mucosal injury and impair the healing process of the damaged mucosa 21 22. However, host response to the infection and environmental factors are thought to be involved in the pathogenesis of H. pylori- related gastroduodenal disease 4 *‘. In particular, an increase in the proliferative activity of gastric epithelial cells without a corresponding increase in apoptosis has been suggested to be implicated in H. pylori-related gastric carcinogenesis 23. EGF-related growth factors play a crucial role in the maintenance of gastric homeostasis s. In particular, they are involved in the protection and healing of the gastric mucosa 6-8 and modulate the balance between proliferation and apoptosis in normal and damaged GI mucosa s ‘). In this study, we evaluated the expression of HB-EGF and AR, members of the EGF receptor family of ligands, in the antral gastric mucosa obtained from dyspeptic patients with and without H. pylori infection and found a significant increase in the gastric tissue levels of both peptides in H. pylori-infected subjects compared with non-infected patients. In H. pylori-infected subjects we found an approximately 2.5- fold increase in HB-EGF and a 2-fold increase in AR mRNA expression compared with non-infected patients with and without endoscopic or histological gastric damage. Also, HB-EGF and AR immunostaining appeared more intense in the H. pylori-infected gastric mucosa than in the non-infected mucosa. In H. pylori- infected subjects, the immunostaining for HB-EGF and AR was detectable not only in the superficial epithelium but also deeper in the glandular compartment of the gastric mucosa. We also observed a number of cells showing nuclear immunoreactivity for AR in the glandular compartment of H. pylori-infected gastric mucosa but not in the non-infected mucosa. Nuclear AR immunoreactivity has previously been reported in a number of studies, but whether AR plays any biological role in the nucleus is still uncertain l4 2s. Nuclear localization of growth factors and their receptors has now been reported for many molecules and suggested to play a role in intracellular signalling 26. Whether nuclear AR immunoreactivity in H. pylori-infected gastric mucosa has any biological significance needs to be determined. In agreement with our previous report showing that H. pylori-induced up-regulation of HB- EGF and AR in gastric cells in vitro was independent of CagA, we did not detect any difference in HB-EGF

or AR mRNA levels between the seven patients with, and the three patients without anti-CagA antibodies. However, the role of the Cag status in HB-EGF or AR up-regulation in H. pylori-infected subjects needs further investigation. Whether a relationship exists between H. pylori infection and EGF-related peptides is not completely clear. TGF-a expression has been reported to be decreased both at the mRNA and protein level in H. pylori gastritis in humans l4 Is. We have recently shown that the expression of HB-EGF and AR, but not TGF-a, is up- regulated in human gastric epithelial cells exposed to H. pylori in vitro . I3 Also, as in our study, an increase in AR mRNA expression in the antrum but not the body of patients with H. pylori gastritis independent of the tag status has recently been demonstrated by Cox et al. *‘. On the other hand, a decrease in HB-EGF and AR mRNA expression has been reported to occur in H. pylori-infected human gastric mucosa “. The apparent discrepancy between our study and the study of Schie- mann et al. Is showing down-regulation of HB-EGF and AR in H. pylori gastritis might be due to the fact that they studied 7 H. pylori-infected patients all with duodenal ulcer whereas in our study only 1 of the 10 H. py- Zori-infected patients had duodenal ulcer. Interestingly, the lowest level of HB-EGF and AR mRNA expression in our series of H. pylori-infected subjects was observed in the patient with duodenal ulcer. It is well known that H. pylori infection exerts diverse effects on gastric physiology and this may be associated with different clinical outcomes 28. That H. pylori-infected subjects with non ulcer dyspepsia have a high level of expression of HB-EGF and AR compared with duodenal ulcer patients may suggest a different perturbation of gastric homeostasis in different clinical settings. Human gastric mucosa colonised by H. pylori shows an increased mitogenic activity (30, 31). We found a slight, but significant, increase in gastric epithelial cell proliferation, consistent with similar reports of previously published studies 7-9 30. The increase in mitogenic activity strongly correlated with the increased expression of HB-EGF and AR. Since EGF receptor ligands stimulate gastric epithelial cell mitogenesis and inhibit apoptosis 5 9, our findings suggest that the increased proliferative activity of H. pylori-infected gastric mucosa might be driven by a sustained overexpression of EGF-related growth factors. However, the biological relevance of the small increase in cell proliferation observed in our study needs to be interpreted with cau- tion. This finding contrasts with our previous observa- tion in vitro showing that H. pylori-induced overexpression of HB-EGF and AR in MKN 28 cells is associated with inhibition of cell proliferation 13. One possible explanation for this apparent discrepancy may be that in vitro experimental studies are representative of

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HA-EGF and AR expression in H. pykwi gastritis

an acute H. pylori-mediated effect whereas clinical studies on human subjects infected by H. pylori are representative of the effect of persistent H. pylori infection. Other factors may be involved in the hyperproliferative activity of gastric epithelium in H. pylori gastritis. Peek et al. have recently shown that in H. pylori- colonised Mongolian gerbils, the increased gastric epithelial cell growth may be mediated by gastrin-dependent mechanisms 3’. Interestingly, in insulin-gas&in transgenic mice, hypergastrinaemia is associated with overexpression of HB-EGF 32. Also, gastrin has been demonstrated to up-regulate HB-EGF and AR, but not TGF-a, expression in rat gastric epithelial cells in vitro 33 34. Moreover, H. pylori infection up-regulates the expression of cycle-oxygenase-2 (COX-2), the inducible isoform of the enzyme responsible for prostaglandin synthesis 35, in gastric mucosal cells in vitro 36 and in vivo I7 37 38, the highest level of expression being in the antral mucosa 38. COX-2 also stimulates cell growth and inhibits apoptosis 39 and may, therefore, contribute to the hyperproliferative response of gastric mucosa to H. pylori colonisation. In conclusion, we have shown that H. pylori gastritis is associated with up-regulation of HB-EGF and AR expression. The increased HB-EGF and AR expression strongly and significantly correlates with the increased proliferative activity of the H. pylori-infected gastric mucosa. The identification of H. pylori specific signalling pathways leading to the induction of cell cycle mediators throw further light on the mechanism of H. pylori-associated gastric carcinogenesis and might be of interest for therapeutic purposes to overcome H. py- Zori-associated gastric cancer. However, one must be cautious in drawing conclusions on the pathogenic implications, due to the fact that 5 subjects in the H. pylori-infected group had gastroduodenal erosions at endoscopy and might hypothetically represent a duodenal ulcer phenotype which should be protected against gastric cancer . *l We postulate that H. pylori-associated gastric mucosal cell injury triggers the compensatory overexpression of growth factors which stimulate cell proliferation and inhibit apoptosis s 9. A sustained growth factor overexpression causes an overall increase in the gastric epithelial cell turnover that, together with increased oxidative DNA damage 4o 4’, may ultimately favour the progression from chronic gastritis to adenocarcinoma in the multistep model of gastric carcinogenesis. One may envisage the chemopreven- tive use of a specific EGF receptor tyrosine kinase inhibitor, alone or in combination with a selective COX- 2 inhibitor4*, in those H. pylori-infected subjects who are resistant to conventional therapeutic regimens and potentially at risk for developing adenocarcinoma of the distal stomach.

List of abbreviations

AR: amphiregulin; COX-2: cyclooxygenase-2; EGF: epidermal growth factor; GAPDH: glyceraldehyde3phosphate dehydrogenase; GI: gastrointestinal; H. py/ori: He/icobactarpy/ori; HE-EGF: heparin binding- epidermal growth factor; PBS: phosphate buffer solution; PCNA: proliferating cell nuclear antigen; PCR: polymerase chain reaction: rh: recombinant human; FIT-PCR: reverse transcriptase-polymerase chain reation; TGF-a: transforming growth factor alpha.

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