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B-Raf/Rap1 signaling, but not c-Raf-1/Ras, induces the histidine decarboxylase promoter in Helicobacter pylori infection¹

SILJA WESSLER, ULF R. RAPP^*, BERTRAM WIEDENMANN, THOMAS F. MEYER, TORSTEN SCHÖNEBERG, MICHAEL HÖCKER and MICHAEL NAUMANN²

Max-Planck-Institut für Infektionsbiologie, Abt. Molekulare Biologie, Berlin;
^* Institut für Medizinische Strahlenkunde und Zellforschung, Universität Würzburg;
Medizinische Klinik mit Schwerpunkt Hepatologie und Gastroenterologie, Universitätsklinikum Charité, Campus Virchow-Klinikum, Humboldt Universität Berlin; and
Institut für Pharmakologie, Freie Universität Berlin, Germany

²Correspondence: Max-Planck-Institut für Infektionsbiologie, Abteilung Molekulare Biologie, Schumannstrasse 21/22, 10117 Berlin, Germany. E-mail: naumann{at}mpiib-berlin.mpg.de

SPECIFIC AIMS

Histidine decarboxylase (HDC) is the key enzyme for gastric histamine synthesis; enhanced HDC expression is critically involved in the pathogenesis of gastric disorders, including gastroduodenal ulcer disease. The human gastric microbial pathogen Helicobacter pylori activates the HDC promoter, which can influence the outcome and progress of gastroduodenal diseases. The aim of the present study was to analyze the H. pylori pathogenicity mechanism leading to host cell signaling and activation of the HDC gene.

PRINCIPAL FINDINGS

1. H. pylori-induced HDC promoter activity involves B-Raf and Rap1
We have previously shown that H. pylori infection of gastric AGS cells leads to activation of the kinases ERK and MEK that regulate HDC promoter activity. Possible upstream activators of MEKs are represented by c-Raf-1 and B-Raf. To further elucidate the upstream mechanisms, we analyzed the kinase activity of B-Raf and c-Raf-1 after H. pylori infection. In AGS cells, H. pylori infection resulted in significant activation of the phosphorylation activity of immunoprecipitated B-Raf toward MEK1 substrate within 5 min (Fig. 1 A), whereas c-Raf-1 was not activated (Fig. 1C ). As controls, 8-CPT-cAMP induced B-Raf kinase activity within 1 to 10 min (Fig. 1B ), and cells treated with FCS showed activation of c-Raf-1 (Fig. 1D ). Transfection of B-Raf antisense oligonucleotides into cells carrying a luciferase reporter construct driven by the HDC promoter led to suppression of H. pylori-induced HDC promoter activity. These data indicate that the H. pylori-induced HDC promoter activation strongly depends on B-Raf activation.

View larger version (60K): [in this window] [in a new window]   Figure 1. Selective activation of B-Raf induced in H. pylori infection. AGS cells were infected with A) H. pylori (P12) at a MOI 200 or incubated with B) 100 µM 8-CPT-cAMP for different periods. B-Raf was immunoprecipitated using an anti-B-Raf antibody and the immunocomplexes were analyzed in in vitro kinase assays using MEK1(K97M) as a substrate. c-Raf-1 activation was analyzed by precipitating c-Raf-1 from cells infected with C) H. pylori (P12) or in cells treated with D) 10% FCS. In vitro kinase assays were performed as described above. Aliquots of the immunoprecipitated proteins were separated by SDS-PAGE and blotted; the immunoblots were probed with either anti-B-Raf or anti-c-Raf-1 antibodies to show similar protein amounts in all lanes.

To analyze whether H. pylori induces a direct interaction between B-Raf and the GTPases Rap1 or Ras, we performed coimmunoprecipitation studies. Immunoprecipitation of Rap1 (Fig. 2 A) in H. pylori-infected or 8-CPT-cAMP-treated AGS cells showed an inducible interaction between B-Raf and Rap1. In contrast, the bacterial N-formyl-peptide fMLP did not induce B-Raf/Rap1 interaction. Association of B-Raf and Rap1 was also observed using an anti-B-Raf antibody for immunoprecipitation. In contrast to the B-Raf-Rap1 interaction, no association between c-Raf-1 and Rap1 was observed (Fig. 2B ). Physical interaction between c-Raf-1 and Ras was also observed in FCS-stimulated AGS cells (Fig. 2C ). No interaction between B-Raf or c-Raf-1 and Ras was observed in H. pylori infection. Further, H. pylori infection resulted in a strong activation of Rap1 within 5 min whereas Ras was not significantly activated, indicating that Rap1 represents the predominant upstream activator of B-Raf and MAP kinases in H. pylori-infected cells.

View larger version (25K): [in this window] [in a new window]   Figure 2. Rap1 interacts specifically with B-Raf in response to H. pylori infection. A) AGS cells were infected with H. pylori (P12) for 30 min and incubated with 8-CPT-cAMP for 5 min or fMLP for 30 min; after preparation of cell lysates, Rap1 was immunoprecipitated using an anti-Rap1 antibody (upper panel). B-Raf95kDa was immunoprecipitated using the anti-B-Raf antibody (middle panel). The immunocomplexes were separated by SDS-PAGE and blotted on membranes. The blots were probed with either an anti-B-Raf antibody or an anti-Rap1 antibody. B) AGS cells were infected with H. pylori (P12) for 30 min, then incubated with 8-CPT-cAMP for 5 min or with fMLP for 30 min; after preparation of cell lysates, Rap1 was immunoprecipitated using an anti-Rap1 antibody. The immunocomplexes were separated by SDS-PAGE and blotted on membranes. The blot was probed with an anti-c-Raf-1 antibody. C) AGS cells were infected with H. pylori (P12) for 30 min or treated with 10% FCS for 20 min. Ras was immunoprecipitated using an anti-Ras antibody and interacting proteins were analyzed with an anti-B-Raf or an anti-c-Raf-1 antibody. In all cases, aliquots of the cell lysates were analyzed in immunoblots to show equal protein amounts of Rap1, B-Raf, and c-Raf-1.

2. H. pylori-induced cAMP accumulation is a prerequisite for activation of the HDC promoter
Analyzing whether cAMP, calcium, or protein kinase C (PKC) represent upstream activators of Rap1 involved in the H. pylori-induced signal transduction, these signaling pathways were blocked using specific pharmacological inhibitors. H. pylori-induced Rap1 and ERK1/2 activation was significantly blocked by the adenylate cyclase inhibitor DDA, but not after pretreatment with BIM or BAPTA/AM. Stimulation of cAMP-induced HDC promoter activity was inhibited in cells cotransfected with dominant negative ERK constructs. To investigate whether activation of PKA was involved in HDC activation downstream of cAMP, we analyzed PKA activation in response to H. pylori infection. H. pylori infection of AGS cells led to activation of PKA kinase activity, but neither Rap1 nor ERK activation was abolished in cells treated with the specific PKA inhibitor PKI. H. pylori-induced HDC promoter activity was also independent of PKA activation. These data suggest the involvement of cAMP in activation of the HDC promoter in response to H. pylori infection, but exclude a potential role for PKA.

3. Activation of H. pylori-induced promoter activity is mediated by G_S
We analyzed the role of the heterotrimeric G-proteins G_S and G_i, which are known to stimulate activation of Rap1 signal transduction. Since many G_S-coupled receptors also couple to G_i, we analyzed the role of G_i using pertussis toxin (PTX), which uncouples G_i from its receptor and blocks the signaling. The H. pylori-dependent cAMP accumulation in AGS cells was PTX insensitive, but was affected in cholera toxin (CTX) -treated cells, which leads to G_S down-regulation. G_S depletion by CTX, but not PTX treatment, reduced Rap1 and ERK1/2 activation in H. pylori-infected cells. To support our hypothesis that G_S mediates the signal transduction leading to activation of the HDC promoter in H. pylori infection, we analyzed the effect of PTX and CTX in a transactivation study. In contrast to PTX, we observed in CTX-treated cells a clear suppression of H. pylori-induced HDC activation.

4. H. pylori-released molecule(s) induce the HDC promoter
To further define the mechanism of host/pathogen interaction, we analyzed various isogenic mutants of H. pylori regarding their ability to induce the eukaryotic signal transduction pathway leading to HDC promoter activation. All isogenic H. pylori strains used induced cAMP accumulation, Rap1 and ERK activation, and transactivated the HDC promoter. Using a filter assay, we found that H. pylori released a factor(s) drive(s) independent of bacterial cell contact, the HDC promoter activation. Characterization of the H. pylori released factor(s) by centrifugation tubes, which allow separation by molecular mass revealed a significant activation of the HDC promoter in a fraction containing molecules < 3 kDa. Further separation of the 3 kDa fraction in a Superdex peptide column displayed two fractions that induced HDC promoter activity and ERK1/2 phosphorylation. The HDC stimulatory component(s) found in these fractions could represent a molecule(s) of 1 kDa.

CONCLUSIONS

We defined the pathogenicity mechanism underlying the activation of the HDC promoter in H. pylori-infected epithelial cells and identified for the first time an almost complete signaling cascade mediating the effect of the microbial pathogen to a nuclear target gene (Fig. 3 ). Certain stimuli induce HDC promoter activity via ERK kinase, but heterogenicity exists in the proximal signal elements, which direct ERK-dependent HDC induction. It is known that gastrin induces ERK-dependent HDC promoter activity via PKC and c-Raf-1 independently of Ras, whereas oxidative stress induced in AGS cells triggers ERK-dependent HDC promoter activity in a c-Raf-1/Ras-dependent manner. We found that H. pylori targets upstream of ERK/MEK preferentially B-Raf whereas c-Raf-1 was unaffected. We show that Rap1, and not Ras, actually transmits the H. pylori-induced signal transduction pathway. The role of Rap1 is still elusive in this context, but our data show for the first time the involvement of Rap1 in H. pylori-mediated signal transduction pathways of gastric epithelial cells. Rap1 activation is induced by second messengers, including cAMP, calcium, and diacylglycerol. Application of the intracellular calcium chelator BAPTA/AM or the PKC inhibitor BIM did not influence Rap1 and ERK1/2 activation in response to H. pylori infection, suggesting that neither calcium nor PKC represent functionally relevant upstream activators of Rap1 in the H. pylori infection of AGS cells. Functional inhibition of the adenylate cyclase using DDA blocked Rap1 and ERK1/2 activation after H. pylori infection. Thus, H. pylori stimulates Rap1 and ERK1/2 activity via elevation of adenylate cyclase activity, increasing intracellular cAMP levels. It is interesting that neither inhibition of PKA nor interruption of the Ras-dependent signaling pathway resulted in a decrease of H. pylori-induced HDC promoter activity, indicating that Epac and then Rap1 mediate the H. pylori-induced signal transduction.

View larger version (65K): [in this window] [in a new window]   Figure 3. Model of a H. pylori-activated signal transduction pathway leading to activation of the HDC promoter. Activation of the H. pylori-induced HDC promoter includes activation of ERK1/2 and MEK1/2. Independent of c-Raf-1 and Ras, activation of ERK/MEK is mediated by the small GTPase Rap1 via B-Raf. Proximal signal elements involve cAMP accumulation induced by GS. The bacterial effector(s) that activate an unknown receptor type represent an 1 kDa component(s) released by H. pylori. Like gastrin, the H. pylori-dependent signal transduction leading to HDC promoter activity may contribute to gastric disorders.

In contrast to H. pylori-induced AP-1- and NF-B-dependent gene expression, we showed that H. pylori-induced HDC promoter activity did not involve pathogenicity island (PAI) -encoded protein expression, indicating a novel mechanism of interaction between H. pylori and host cells. H. pylori-induced HDC-gene activation requires the temporally coordinated action of host cell signaling components that become activated by a small molecular mass component(s) (1 kDa) released independent of the PAI-encoded type IV secretion machinery. The molecular nature of the H. pylori 1 kDa component(s) that induce(s) the HDC activity is not known, but its/their ability to activate heterotrimeric G_s suggests that they stimulate GPC receptor types. The bioactive H. pylori-molecule(s) could activate epithelial cell signaling apart from the site of bacterial colonization, allowing novel insights into H. pylori pathogenesis. H. pylori represents a highly successful human microbial pathogen that has infected half of the world‘s population, but the majority of infected individuals do not develop serious clinical diseases. Thus, H. pylori infection is an example of evolutionary forces that operated over an extended time, leading to a balanced interaction that allows bacterial survival while preventing excessive harm to the host. Considering that H. pylori initiates cellular processes and host responses that become increasingly independent of its presence, therapeutic intervention strategies for the interference with the cellular dysregulation could be a subject of growing importance.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0766fje; to cite this article, use FASEB J. (January 14, 2002) 10.1096/fj.01-0766fje

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