Helicobacter pylori activates the histidine decarboxylase promoter through a mitogen-activated protein kinase pathway independent of pathogenicity island-encoded virulence factors.

Helicobacter pylori infection of the gastric mucosa is accompanied by an activated histamine metabolism. Histamine plays a central role in the regulation of gastric acid secretion and is involved in the pathogenesis of gastroduodenal ulcerations. Histidine decarboxylase (HDC) is the rate-limiting enzyme for histamine production, and its activity is regulated through transcriptional mechanisms. The present study investigated the effect of H. pylori infection on the transcriptional activity of the human HDC (hHDC) promoter in a gastric epithelial cell line (AGS) and analyzed the underlying molecular mechanisms. Our studies demonstrate that H. pylori infection potently transactivated the hHDC promoter. The H. pylori-responsive element of the hHDC gene was mapped to the sequence +1 to +27 base pairs, which shows no homology to known cis-acting elements and also functions as a gastrin-responsive element. H. pylori regulates the activity of this element via a Raf-1/MEK/ERK pathway, which was activated in a Ras-independent manner. Furthermore, we found that H. pylori-induced transactivation of the hHDC promoter was independent of the cag pathogenicity island and the vacuolating cytotoxin A gene and therefore may be exerted through (a) new virulence factor(s). A better understanding of H. pylori-directed hHDC transcription can provide novel insights into the molecular mechanisms of H. pylori-dependent gene regulation in gastric epithelial cells and may lead to new therapeutic approaches.

encoded by the cytotoxin-associated gene A (cagA) 1 -associated pathogenicity island (PAI) have been considered associated with enhanced pathogenic potential of the bacterium (1, 6 -8). Individuals infected with PAI-positive H. pylori strains display more severe courses of gastric inflammation and gastroduodenal ulcer disease and appear to develop more frequently gastric adenocarcinomas and mucosa-associated lymphoid tissue lymphomas (9,10).
The PAI represents a cluster of 31 genes that encode presumably a specialized secretion system that enables the bacterium to expose or secrete particular proteins that are involved in induction of intracellular signaling cascades of target cells (7). A molecular concept for the enhanced pathogenicity of PAI-positive H. pylori strains has been provided by the observation that genes located within the PAI are indispensable for activation of NF-B and AP-1 as well as stimulation of IL-8 production and secretion in gastric epithelial cells and therefore seem to elicit a more pronounced immune response (7,(11)(12)(13)(14)(15). In addition to cag-related genes, factors outside the PAI also appear to contribute to the differences in the pathogenic potential of H. pylori (16,17).
The gastric inflammatory response to H. pylori is characterized by mucosal infiltration of neutrophils and lymphocytes leading to enhanced release of cytokines and chemokines (3,18). In addition, increased gastric histamine secretion appears to contribute to the inflammatory changes and tissue damage associated with chronic H. pylori infection of the gastric mucosa (19 -21). Histamine represents the major stimulus controlling gastric acid secretion and is produced and secreted by enterochromaffin-like (ECL) cells of the corpus mucosa (22)(23)(24)(25)(26). Histidine decarboxylase (HDC) is the key enzyme for histamine production in gastric ECL cells, and its enzymatic activity is to a large extent regulated through enhanced transcription of the HDC gene (27)(28)(29)(30). In addition to its central role in regulation of gastric acid secretion, increased HDC gene expression has also been found to be associated with gastric inflammation and development of gastroduodenal ulceration (31)(32)(33)(34)(35). Furthermore, we found that oxidative stress, which is commonly increased in ulcerative and inflammatory diseases affecting the gastric mucosa, is capable of transactivating the hHDC promoter in vitro (36). Aside from its ulcerogenic potential, histamine has also been shown to possess immunomodulatory properties in the context of mucosal inflammations, contributes to the healing of ulcerative lesions of the gastric and intestinal mucosa, and has also been shown to stimulate the growth of gastric epithelial cells (37)(38)(39)(40)(41). A current in vivo study demonstrated that H. pylori infection is associated with increased mucosal histamine levels as well as an expansion of the gastric ECL cell lineage (42). The hypothesis that these changes could be at least in part be attributed to a direct effect of H. pylori on ECL cells has been substantiated by the finding that H. pylori can stimulate histamine secretion from isolated rat ECL cells as well as ECL cell proliferation in vitro (43). Although these findings strongly suggested that H. pylori can directly influence the histamine metabolism of gastric ECL cells, potential molecular mechanisms that could underlie this effect are unclear.
It has been demonstrated that in gastric epithelial cells H. pylori infection elevates the abundance of "classical" second messenger molecules such as Ca 2ϩ , cyclic adenosine monophosphate (cAMP), and inositol trisphosphate (44). Although H. pylori has been shown to stimulate phosphorylation of several cellular proteins in gastric cancer cells in vitro (44,45), intracellular signaling cascades activated by the bacterium are largely unknown. In a current study, we investigated the effect of H. pylori on the AP-1 transcription factor complex in gastric cancer cells in vitro and found that AP-1 activity is regulated by H. pylori through activation of c-Jun NH 2 -terminal kinase (JNK) (13), which belongs to the superfamily of "mitogen-activated protein kinases" (MAP kinases) (46,47). In addition to the JNK cascade, the MAP kinase superfamily comprises the extracellular-regulated kinase (ERK) pathway (46). Although some overlap between both pathways has been described (46), the JNK cascade activates primarily transcription factors involved in the "stress response" of eukaryotic cells, whereas the ERK pathway has been linked to genes involved in cellular proliferation and differentiation (46 -48). Recently we demonstrated that ERK-related signaling cascades also play a central role in the transmission of the effects of gastrin and oxidative stress on the hHDC promoter in gastric cancer cells, whereas the JNK pathway is not involved in hHDC gene regulation (36,49). Therefore, it is highly likely that the ERK cascade represents a potential target signaling route through which activators of hHDC gene transcription exert their transactivating effect on the hHDC promoter.
To investigate whether H. pylori can directly influence the transcriptional activity of the hHDC promoter, we performed in vitro studies employing hHDC-luciferase reporter gene constructs as well as various hHDC promoter mutants. Furthermore, we aimed to analyze the signal transduction pathways and nuclear factors responsible for transmission of this effect on the hHDC promoter. Finally, we analyzed the virulence factors involved in regulation of the hHDC promoter by H. pylori.
Bacteria and Infection-The following H. pylori strains were used for infection experiments: P12 strains (wild type) and the isogenic cagA * (cagA with a probable polar effect), vacA (50), and PAI (missing the cag pathogenicity island (PAI); and G27 (wild type) and the isogenic cagI strain (8). For the construction of the PAI strain two approximately 2-kb DNA fragments upstream of the cag-PAI (region 545254 -547164) and downstream of the PAI (584570 -586563) (51) were amplified by polymerase chain reaction and cloned into pBluescript separated by a kanamycin resistance gene. The plasmid was transformed into H. pylori (P12), and one transformant was analyzed by polymerase chain reaction for correct allelic exchange of the PAI with the resistance gene. H. pylori strains were grown on agar plates containing 10% horse serum in a microaerophilic atmosphere (generated by Campy-Gen, Oxoid, Basingstoke, U. K.) at 37°C for 48 -72 h. 24 h after infection H. pylori was harvested in phosphate-buffered saline (pH 7.4), diluted corresponding to the multiplicity of infection (m.o.i.), and incubated with the epithelial monolayer. Infection with H. pylori was monitored routinely by light microscopy.
Electrophoretic Mobility Shift Assay (EMSA)-Nuclear extracts were prepared by using a non-ionic detergent method as described previously (55). For the detection of gastrin-responsive element (GAS-RE) DNA binding activity, equal amounts of nuclear protein extracts were incubated with labeled oligonucleotides containing the ϩ1 ϩ27 GAS-RE binding site sequence of the human HDC promoter: The oligonucleotide containing the GAS-RE recognition site was labeled using T4 kinase (Roche Molecular Biochemicals GmbH, Mannheim, Germany) in the presence of [␥-32 P]ATP. The DNA binding reactions were performed using a binding buffer containing 10 mM Tris (pH 7.5), 2 g of poly(dI-dC), 1 g of bovine serum albumin, 10 mM MgCl 2 , 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, and 4% Ficoll for 20 min at room temperature. For competitions, increasing amounts of an unlabeled oligonucleotide were included in the bandshift reaction. The DNA binding activity of NF-B was performed with an Ig oligonucleotide as described previously (55). The DNA binding reactions were performed using a binding buffer containing 20 mM HEPES (pH 8.4), 60 mM KCl, 5 mM dithiothreitol, 1 g of bovine serum albumin, 2 g of poly(dI-dC), and 10% glycerol for 20 min at 30°C. The reaction products were analyzed by electrophoresis in a 6% polyacrylamide gel using 12.5 mM Tris, 12.5 mM boric acid, and 0.25 mM EDTA (pH 8.3). The gels were dried and exposed to Amersham TM films (Amersham Pharmacia Biotech) at Ϫ70°C using an intensifying screen. To identify the H. pylori-responsive element of the hHDC promoter sufficient for the transcriptional response, we analyzed a series of hHDC 5Ј-deletion constructs (Fig. 1B). Removal of nucleotides from the 5Ј-end down to 125 bp upstream of the Cap site (ϩ1) had no significant influence on the H. pylori-induced luciferase activity. Similar results were obtained in cells treated with PMA (data not shown). Because this region comprises the ϩ1 to ϩ27 bp GAS-RE of the hHDC proximal promoter, we used a luciferase construct in which the GAS-RE sequence hHDC ϩ1 to ϩ27 was ligated upstream of the enhancerless herpes simplex virus 1 thymidine kinase (TK) promoter. We found that this element was capable of conferring H. pylori responsiveness to the same extend as the longest hHDC 5Ј-flanking fragments.

H. pylori Infection Stimulates HDC Promoter Activity through a Minimal 27-Base Pair Promoter Element-AGS
To underline that the GAS-RE is activated by H. pylori through enhanced binding of nuclear factors, we analyzed the DNA binding activity of nuclear proteins to the ϩ1 to ϩ27 sequence in EMSAs. Enhanced binding of AGS nuclear proteins was observed within 30 min postinfection (Fig. 1C, lanes  1-4). Treatment of AGS cells with PMA, a strong inducer of hHDC promoter activity, resulted in a strong increase of DNA binding activity of transcription factors within 10 min (Fig. 1C,  lanes 5-7). Because H. pylori-and PMA-induced complexes produced identical bandshifts it can be concluded that the H. pylori-stimulated complex consists of GAS-RE-binding proteins (GAS-RE-BPs). This was further confirmed by the finding that the complex stimulated by H. pylori could be competed away by an excess of unlabeled hHDC ϩ1/ϩ27 bp oligonucleotide (Fig.  1C, lanes 8 -10). Based on these data, H. pylori was identified to induce very specific (m.o.i. of 5-50) enhanced hHDC promoter activity through activation of the GAS-RE-BP1/2 transcription factors that bind to the ϩ1 to ϩ27 bp minimal element.
Activation of the hHDC Promoter Is Independent of cag-PAIencoded Gene Expression-To investigate the role of cag genes for activation of the hHDC promoter by H. pylori, AGS cells were colonized with different isogenic mutants lacking certain cag genes. AGS cells transfected with the 1.8-kb hHDC-luc construct exerted after infection with H. pylori wild type strains (P12 and G27) a strong increase in luciferase activity ( Fig. 2A, left panel). Similar activation was obtained in AGS cells, infected with isogenic mutants, lacking the vacA, cagA*, and cagI genes. Additionally, the isogenic H. pylori strain PAI, which lacks the entire PAI, potently induced the hHDC promoter activity, supporting the notion of a cag-independent activation of the hHDC promoter by H. pylori. In contrast, in AGS cells transfected with a reporter gene construct in which luciferase expression was under control of the NF-B consensus element of the HIV promoter, H. pylori mutants lacking cagA*-cagI genes or the entire PAI did not stimulate reporter gene expression ( Fig. 2A, right panel). The cag-independent activation of the HDC promoter activity was also investigated by EMSA. Enhanced DNA binding of the GAS-RE-BP1/2 to the HDC enhancer element was observed in AGS cells infected with any H. pylori (Fig. 2B, lane 1-8, left panel). Using the Ig-NF-B oligonucleotide to detect NF-B DNA binding activity, colonization with the H. pylori strains P12, vacA, and G27 induced strong activation of NF-B (Fig. 2B, lanes 1-3 and 6, right panel), whereas the knockout mutants cagA*, cagI, and PAI showed a strongly reduced NF-B DNA binding activity (Fig. 2B, lanes 4, 7, and 8, right panel).
H. pylori-stimulated hHDC Promoter Activity Involves Activation of the ERK/MEK Kinases-To study the signaling that is induced by H. pylori in a cag-independent manner, we investigated the capability of H. pylori to induce activation of certain MAP kinase pathways. Subconfluent monolayers of AGS cells were infected with H. pylori (P12), and cell lysates were prepared after different time points postinfection and analyzed for activated and phosphorylated ERK1/2 and MEK1/2 by Western blot analysis using phospho-specific antibodies. H. pylori activated both ERK1 and ERK2 in AGS cells within 30 min after infection (Fig. 3A, lanes 1-5), comparable to the activation induced by stimulation with PMA (Fig. 3A, lanes  6 -9). Similar results were obtained using phospho-specific antibodies to detect activated MEK1/2. MEK1 and MEK2 were also phosphorylated within 30 min after H. pylori infection or PMA treatment (Fig. 3B, lanes 1-9). To investigate whether the H. pylori-induced activation of the ERK/MEK kinases is cagindependent, we studied the effects of the H. pylori mutants on the activation of ERK1/2 and MEK1/2. In contrast to the AP-1-activating kinase pathway (13) the infection of AGS cells with H. pylori knockout mutants (vacA, cagA, PAI, and cagI) in all cases stimulated activation of ERK1/2 and MEK1/2 ( Fig. 3A  and B, right panel). As a control, we used the same extracts to analyze the H. pylori-infected cells for degradation of the NF-B inhibitor IB␣. Colonization of AGS cells with H. pylori (P12) for different periods of time resulted in decreasing amounts of IB␣ (Fig. 3C, lanes 1-5) as well as treatment of the cells with PMA (Fig. 3C, lanes 6 -8). Corresponding to the NF-B activation (Fig. 2B, lower panel), IB␣ is degraded in response to infection with H. pylori strains P12, vacA, and G27 (Fig. 3C, lanes 10, 11, and 14) but remains unaffected after infection with H. pylori strains cagA*, PAI, and cagI (Fig. 3C,  lanes 12, 13, and 15). These data indicate that H. pylori infec-tion has the capacity to induce ERK/MEK activation independent of H. pylori cag gene expression.
To examine whether these H. pylori-activated MAP kinases are involved in the upstream signaling regulating the hHDC promoter activity, we cotransfected AGS cells with the 1.8-kb hHDC-luciferase construct and dominant negative ERK1 (DNERK1) and ERK2 (DNERK2) constructs. Infection of AGS cells with H. pylori or PMA treatment resulted in a 3-fold induction of the HDC promoter activity compared with nontreated cells (Fig. 4A, left panel). After cotransfection of single inhibitory kinase constructs (DNERK1 or DNERK2), the H. pylori-and PMA-stimulated transactivation activity of the HDC promoter was completely inhibited, just as after expression of both DNERK1 and DNERK2 together. As an additional control we used the AGS-B cell line, which has been stably transfected with the human cholecystokinin-B/gastrin receptor (30). Cells exposed to gastrin reacted with a strong increase of the hHDC promoter transactivation activity after 12 h, which was diminished by expression of DNERK1 and DNERK2 mutants (Fig. 4A, left panel). The selective effect of the DNERK1 and DNERK2 kinase constructs was demonstrated using a Studying the H. pylori mutants, AGS cells were infected for 30 min (right panels). Cells were lysed, and 30 g of lysates were separated by SDS-polyacrylamide gel electrophoresis and blotted onto membranes. Phosphorylation of ERK1/2 (A) and MEK1/2 (B) was detected using phospho-specific antibodies in Western blot analysis (upper panels). As a loading control, the same amounts of the cell lysates were blotted and probed with non-phospho-specific anti-ERK2 and anti-MEK1 antibodies (lower panels). Additional bands, beside ERK2 and MEK1, observed in some lanes represent a cross-reactivity with other antigens recognized by the antibodies. C, as an additional control we determined the IB␣ abundance in response to H. pylori colonization using an IB␣ antibody for immunodetection. The positions of the recognized proteins are indicated. luciferase construct driven by a HIV-NF-B enhancer element. In contrast to the PMA-induced NF-B transactivation, overexpression of dominant negative mutants of ERK1 and ERK2 did not block the H. pylori-induced NF-B transactivation (Fig.  4A, right panel). These data indicate a strong and selective involvement of ERK kinases in the HDC promoter-activating pathway.
To block MEK activation in response to H. pylori colonization, we preincubated the AGS cells with the MEK-selective inhibitor PD98059. Pretreatment of AGS cells for 30 min with 50 M PD98059 resulted in a total inhibition of the hHDC promoter transactivation activity induced by H. pylori, indicating the involvement of MEK in the upstream signaling of the hHDC promoter activity (Fig. 4B, left panel). The selectivity of the targeted kinases was also investigated using a HIV-NF-B luciferase construct. The H. pylori-induced NF-B transactivation was not affected by PD98059 pretreatment (Fig. 4B, right  panel).
Raf-1-dependent Activation of the hHDC Promoter in Response to H. pylori Colonization-Possible upstream activators of MEK1/2 are represented by molecules like Raf-1 or MEKK1 (MEK kinase 1) (46). Cells expressing a DNRaf-1 construct and infected with H. pylori or treated with PMA or gastrin were analyzed for transactivation activity of the hHDC promoter. Compared with mock-transfected cells, the hHDC promoter transactivation activity was strongly reduced (Fig. 5A, left panel), whereas expression of DNRaf-1 had no influence on the activation of NF-B induced by H. pylori (Fig. 5A, right panel). These data lead to the suggestion that the Raf-1 kinase lies upstream in the specific signal pathway leading to MEK/ERKdirected activation of the hHDC promoter. The possible role of MEKK1 in the H. pylori-induced activation of the hHDC promoter was investigated using constructs expressing DNMEKK1. Colonization of transiently transfected AGS cells with H. pylori or treatment with PMA induced hHDC promoter activity that was not affected by overexpression of dominant negative MEKK1 (Fig. 5B, left panel). To show the functional dominant negative effect of the MEKK1 construct, we cotransfected the DNMEKK1 cDNA with the AP-1 luciferase construct. Expression of DNMEKK1 inhibited the EGF-induced AP-1 activation (Fig. 5B, right panel).
Activation of the H. pylori-induced hHDC Promoter Activity Based on a Ras-independent Signaling-The small G-protein Ras is one known upstream regulator of Raf-1. In the following we studied whether Ras activation contributes to the stimulation of the hHDC promoter activity. To explore the capacity of Ras to induce H. pylori-mediated hHDC promoter activity, we tested if dominant negative Ras (DNRas15 and DNRas17) blocks activation of the hHDC promoter. In contrast to the inhibition by DNERKs and DNRaf-1, neither DNRas15 nor DNRas17 blocks the H. pylori-induced hHDC promoter activity (Fig. 6, left panel), whereas the EGF-induced AP-1 transactivation activity was inhibited significantly by expression of DNRas15 and DNRas17 (Fig. 6, right panel). DISCUSSION In the present study we demonstrated that colonization of a permanent gastric epithelial cell line with H. pylori stimulates the transcriptional activity of the hHDC promoter in vitro. Furthermore, we found that the transactivating effect of H. pylori is independent of the vacA gene and genes encoded by the cag-associated pathogenicity island, which have been implemented in more severe clinical outcomes of chronic gastric H. pylori infections (1,7,8). The analysis of cis-and transactivating factors involved in H. pylori-dependent hHDC regulation revealed that a proximal element at ϩ 1 to ϩ27 bp, which has been identified previously to be responsible for gastrindependent regulation of the hHDC gene, is also mediating the effect of H. pylori on the hHDC promoter (53,54).
H. pylori has been shown to stimulate the expression of proinflammatory cytokines such as IL-1␤, IL-6, tumor necrosis factor-␣, and IL-8 (18,19). Studies about H. pylori-induced IL-8 secretion (11,14,15) demonstrated that activation of NF-B is a central mechanism through which H. pylori transactivates the IL-8 gene promoter, whereas the H. pylori-dependent transactivation of the hHDC promoter does not involve NF-B. The proximal hHDC ϩ1 to ϩ27 element does not display any homology to known transcription factor binding sites, and competition studies with oligonucleotides representing consensus binding elements of various known transcription factors demonstrated that this element is not bound by NF-B or other well characterized nuclear factors such as AP-1, Sp1, or CREB (53). The view that H. pylori-dependent transactivation of the hHDC promoter does not require NF-B is substantiated further by the finding that isogenic H. pylori mutants (cagA, PAI, cagI), which were not able to transactivate a NF-B-luciferase reporter gene construct, stimulated the binding of nuclear proteins to the hHDC ϩ1 to ϩ27 element and transactivated a hHDC 1.8-kb luciferase reporter construct in AGS cells. Therefore, it can be concluded that the hHDC ϩ1 to ϩ27 bp element represents a new nuclear target sequence of H. pylori through which the bacterium can influence gene expression independent of vacA-or PAI-encoded genes in gastric epithelial cells (Fig. 7).
Previous analysis of the nuclear proteins regulating the hHDC ϩ1 to ϩ27 bp element demonstrated that it is bound by two so far unknown transcription factors (54). These proteins with a molecular size of 35 and 52 kDa, respectively, were termed GAS-RE-BPs because their binding to the ϩ1 to ϩ27 element is indispensable for full gastrin responsiveness of the hHDC promoter (54). Similar to gastrin and PMA, H. pylori infection of AGS cells stimulated binding of nuclear proteins to this element. EMSA analysis of H. pylori-and PMA-stimulated AGS cells demonstrated that the nuclear proteins stimulated by both factors displayed identical bandshifts. Additionally, the H. pylori-induced complex binding to the hHDC ϩ1 to ϩ27 sequence could be competed away with an excess of cold ϩ1 to ϩ27 bp oligonucleotide. Therefore, it appears very likely that the transcription factors binding to the ϩ1 to ϩ27 bp element in response to H. pylori represent the previously described GAS-RE-BPs. Because the GAS-RE-BPs represent two novel transcription factors, further analysis of these factors may lead to the identification of genes that have so far not been linked to the epithelial response to H. pylori. Further, our findings indicate that in addition to the well characterized vacA and cag genes, H. pylori expresses (a) virulence factor(s), which enable(s) the bacterium to activate alternative target genes. The fact that this/these factor(s) appear to be independent of genetic regions that have been associated with enhanced pathogenic potential of H. pylori strains indicates that additional gene loci outside the so far characterized virulence factors may contribute to the overall pathogenesis of H. pylori on the gastric mucosa.
To understand the molecular pathways by which the effect of H. pylori colonization of AGS cells is transmitted into the nucleus, we analyzed signal transduction cascades involved in H. pylori-dependent hHDC transactivation. We found that the transactivating effect of H. pylori is transmitted via a signaling cascade comprising ERKs and their upstream activating kinases MEK and Raf-1, respectively. ERKs and MEKs are activated by H. pylori colonization with a similar time course showing peak phosphorylation after 30 min of infection. In contrast to the effect of H. pylori on the hHDC promoter, activation of NF-B and AP-1 by the bacterium strictly required cag gene expression (13). Therefore, the virulence factor(s) underlying H. pylori-dependent activation of Raf-1/MEK/ ERK-dependent signaling cascades resulting in enhanced HDC transcription appear(s) to act independently of the bacterial factors controlling the acute cytokine response featuring activation of stress response signaling pathways and NF-B activation (Fig. 7). Our results indicate that different virulence factors of H. pylori are capable of activating distinct branches of the MAP kinases signaling system, which appear to result in transactivation of different epithelial target genes. This observation further suggests that dependent on the virulence factors expressed, H. pylori strains may be able to elicit a differential epithelial signaling response, which may lead to transactivation of a specific set of genes.
A recent study described that incubation of KATO III gastric carcinoma cells with H. pylori supernatants resulted in inhibition of ERK-signaling stimulated by EGF and that this effect was vacuolating toxin A-dependent (56). In contrast to this study, we found that exposure of AGS cells to two different strains of intact H. pylori (P12 and G27) resulted in enhanced MEK/ERK phosphorylation. Furthermore, in contrast to the findings by Pai et al. (56), the effect of H. pylori on the MEK/ ERK cascade was vacuolating toxin A-independent. Although we did not investigate the effect of H. pylori on EGF-stimulated ERK signaling in the AGS model, the robust ERK/MEK phos- phorylation in response to intact H. pylori demonstrates that the bacterium is capable of stimulating this mitogenic pathway. Activation of the MEK-1/ERK cascade by classical mitogens such as EGF is typically induced through activation of Ras and Raf-1 (46). Because application of two different dominant negative Ras mutants did not influence the transactivating effect of H. pylori on the HDC promoter, the functional involvement of Ras in this context is unlikely. Previous studies from our laboratory demonstrated that alternatively to the Ras/ Raf-1 sequence, the MEK/ERK cascade in AGS cells can also be activated through a Ras-independent, protein kinase C/Raf-1dependent pathway (49). Whether protein kinase C-dependent signaling events are involved in the coupling of the Raf/MEK/ ERK cascade to upstream signaling events triggered by H. pylori is currently under investigation in our laboratory.
Based on the clinical data currently available, the exact impact of H. pylori-stimulated HDC transcription on the overall pathophysiology of H. pylori in the stomach is currently unclear and has to be further clarified in in vivo studies. It can be speculated that dependent on the onset, duration, and/or intensity of activation, enhanced H. pylori-stimulated hHDC transcription could contribute to either tissue damage or processes of mucosal restitution and repair in the stomach (32,33,37,38). Furthermore, a direct effect of H. pylori on HDC gene expression resulting in enhanced histamine production and secretion could help to maintain an acidic gastric environment, favoring the survival of H. pylori over non-acid-resistant bacteria. Overall, H. pylori-dependent transactivation of the hHDC gene represents a new molecular model for the interaction of the bacterium with gastric epithelial cells. Our data for the first time demonstrate that H. pylori is capable of activating a gastric target gene through an ERK-dependent signaling pathway, independent of vacuolating toxin A-and PAI-related virulence factors. Therefore, a detailed analysis of the molecular mechanisms underlying the effect of H. pylori on the hHDC gene could contribute to a better understanding of the molecular pathogenesis of H. pylori infections and could probably lead to new approaches for the treatment of H. pylori-associated gastric diseases.