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Helicobacter pylori stimulates host vascular endothelial growth factor-A (vegf-A) gene expression via MEK/ERK-dependent activation of Sp1 and Sp3¹

MATHIAS Z. STROWSKI, THORSTEN CRAMER, GEORGIA SCHÄFER, STEFAN JÜTTNER, ANNA WALDUCK^*, ERNESTINA SCHIPANI, WOLFGANG KEMMNER, SILJA WESSLER, CHRISTIAN WUNDER^*, MATTHIAS WEBER^*, THOMAS F. MEYER^*, BERTRAM WIEDENMANN, THOMAS JÖNS^*, MICHAEL NAUMANN and MICHAEL HÖCKER²

Medizinische Klinik mit Schwerpunkt Hepatologie und Gastroenterologie, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, Berlin;
^* Max-Planck-Institut für Infektionsbiologie, Berlin;
Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA;
Robert-Rössle-Klinik und Max-Delbrück-Centrum, Berlin;
Institut für Experimentelle Innere Medizin, Otto-von-Guericke-Universität, Magdeburg; and *Institut für Anatomie, Charité-Universitätsmedizin Berlin, Campus Mitte, Berlin, Germany

²Correspondence: Medizinische Klinik, mit Schwerpunkt Hepatologie und Gastroenterologie, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail: hoecker{at}charite.de

SPECIFIC AIMS

Vascular endothelial growth factor-A (VEGF-A) is a key regulator of inflammatory and tumor-associated angiogenesis. Helicobacter pylori (H. pylori) plays a critical role in the pathogenesis of benign and malignant gastric diseases. It has been suggested that similar to other chronic inflammatory disorders, H. pylori infection is associated with activation of host angiogenesis, but the underlying mechanisms and participating angiogenic growth factors have not been identified. Therefore, we investigated the influence of the bacterium on vegf-A as a candidate host target gene in gastric H. pylori infection.

PRINCIPAL FINDINGS

1. H. pylori infection up-regulates vegf-A mRNA and protein abundance in gastric epithelial cells and stimulates vegf-A gene promoter activity
To determine the influence of H. pylori on vegf-A gene expression, we used in vitro and in vivo infection models. Exposure of permanent AGS gastric epithelial cells to the bacterium led to enhanced production and secretion of VEGF-A protein (Fig. 1 A). Similarly, H. pylori potently stimulated vegf-A mRNA in AGS cells (Fig. 1B ) as well as in epithelial cells of the murine stomach, strongly suggesting that enhanced transcription is the major mechanism mediating H. pylori-triggered vegf-A regulation (Fig. 1C, D ). Accordingly, the activity of a vegf-A promoter reporter gene construct (-2018hvegf-A-luc) transfected into gastric epithelial cells was potently stimulated by the bacterium in a dose- and time-dependent manner (3- to 4-fold). To evaluate a potential contribution of oxidative stress in transmission of H. pylori-dependent effects on the vegf-A gene, we used the antioxidant N-acetylcysteine (ACC) in transfection studies. We found that ACC potently inhibited the stimulating effect of the model oxidant H₂O₂ on vegf-A promoter activity but did not influence H. pylori-triggered vegf-A transactivation, confirming that oxidative stress pathways are not involved in H. pylori-dependent vegf-A regulation. To identify the regulatory promoter element(s) mediating the effects of H. pylori, we performed progressive deletion analysis of the vegf-A 5'-flanking sequence in functional transfection assays and found that a region spanning -85/-50 is indispensable for H. pylori-dependent regulation of the vegf-A gene (Fig. 2 A). A single copy of the -85/-50 element was able to confer H. pylori responsiveness to the per se H. pylori-insensitive, heterologous thymidine kinase (TK) promoter system pT81 (Fig. 2B ), further highlighting the functional importance of this element for H. pylori-dependent vegf-A gene regulation.

View larger version (57K): [in this window] [in a new window]   Figure 1. H. pylori infection stimulates vegf-A gene expression in gastric epithelial cells. A) AGS gastric cancer cells exposed to H. pylori for 6 h were assayed for cellular VEGF-A production or VEGF-A release into cell culture supernatants. Results are expressed as mean ±SE of 3 separate experiments. Asterisks indicate statistically significant differences (***P<0.001). B) After H. pylori infection, vegf-A and ß-actin mRNA levels were determined by duplex RT-PCR in AGS cells. Data represent a typical result obtained from a series of 3 independent experiments. C) Mice were infected with mouse-adapted H. pylori strain Hp76 or received PBS. RNA was prepared from the mucosa, then analyzed by real-time PCR. All experiments were repeated at least twice and the data represent a typical result. D) vegf-A in situ hybridization on histological sections prepared from stomachs of a sham-infected (a, b) or H. pylori-infected (c, d) mouse. Tissues were obtained 24 h after infection and processed. Representative bright- (HE staining; a, c) and darkfield (b, d) images show corresponding regions of a mouse stomach.

View larger version (31K): [in this window] [in a new window]   Figure 2. Identification of the H. pylori-responsive element of the vegf-A gene promoter. AGS cells transfected with vegf-A 5'-deletion constructs were infected with H. pylori or treated with PBS. After 4 h, cells were harvested and lysates were analyzed for luciferase activities. B) The vegf-A -85/-50 element confers H. pylori sensitivity to a heterologous reporter gene system. AGS cells were transiently transfected with construct vegf-A(-88/-50)-luc or corresponding empty vector pT81-luc, then exposed to H. pylori, PMA [10 nM], or PBS (control). After 4 h, cells were harvested and lysates were analyzed for luciferase activities. Data represent mean ±SE obtained from 3 independent experiments; statistically significant differences (*P<0.05; ***P<0.001).

2. H. pylori stimulates vegf-A transcription via activation of Sp1 and Sp3 at proximal GC-rich elements
For further insight into the molecular mechanisms underlying H. pylori-triggered vegf-A gene expression, transcription factors mediating effects of the bacterium on the H. pylori-responsive element -88/-50 were characterized. Two major protein:DNA complexes were identified at the -88/-50 site; H. pylori infection time-dependently stimulated the intensity of both complexes, reflecting increased binding of transcription factors to the vegf-A gene promoter. Detailed EMSA competition and supershift studies identified complex I to represent Sp1 protein whereas complex II was shown to consist of Sp3. Functional assays using Gal4-Sp1 and Gal4-Sp3 expression constructs along with a 5xGal4-Luc reporter plasmid showed that H. pylori infection also potently stimulated the transactivating capacity of both transcription factors (Sp1: twofold; Sp3: fourfold). Detailed mutational analysis of the vegf-A -88/-50 element in functional assays and EMSA experiments demonstrated that three GC-boxes located within the -88/-50 sequence function as binding sites of Sp1 and Sp3 and that the two most 5' located GC-boxes at -73/-66 and -58/-52 were indispensable for basal and H. pylori-triggered vegf-A transcription. The distal GC-rich element at -85/-77 was found to be of no functional importance for H. pylori-dependent effects on the vegf-A gene promoter.

3. H. pylori regulates the vegf-A gene through stimulation of MEK/ERK1/-2 host signaling pathways
As earlier work identified MEK/ERK and JNK cascades as important signaling pathways mediating H. pylori effects on host target genes, we investigated the role of these kinase cascades for H. pylori-triggered vegf-A regulation. Wild-type H. pylori as well as isogenic mutants lacking cagPAI potently stimulated MEK1/-2 and ERK1/-2 phosphorylation. A cagPAI-deficient H. pylori strain was unable to stimulate JNK phosphorylation, implying that JNK pathways do not contribute to H. pylori-dependent regulation of vegf-A gene transcription. Inhibition of the ERK1/-2 pathway by transfection of appropriate dominant-negative ERK1/-2 mutants abrogated H. pylori-dependent vegf-A transactivation. In contrast, interruption of the MKK4/JNK pathway by application of a dominant-negative MKK4 mutant had no effect on H. pylori-dependent vegf-A regulation, but blunted the effect of the bacterium on the AP-1-regulated reporter gene construct 4xTRE-luc. To further confirm the functional relevance of the MEK/ERK-related signaling cascade for vegf-A gene regulation, we overexpressed MEK1, ERK1, and/or ERK2 together with the (-2018)vegf-A promoter reporter gene construct in AGS cells and found that the vegf-A promoter is highly responsive to kinases of the MEK>ERK1/-2 signaling module.

CONCLUSIONS AND SIGNIFICANCE

Our study demonstrates for the first time potent up-regulation of host VEGF-A protein and mRNA levels in response to H. pylori exposure and shows that these effects are accompanied by potent transactivation of the vegf-A promoter, suggesting that enhanced transcription of the vegf-A gene represents the underlying mechanism. Angiogenesis, the development of new blood vessels from existing endothelial precursors, is a pathophysiological mechanism involved in the pathogenesis of inflammatory and ulcerative epithelial lesions as well as malignant tumor growth and metastasis. VEGF-A is one of the most potent proangiogenic stimuli of neoangiogenesis. Enhanced vegf-A gene expression has been linked to healing of peptic lesions and to growth and metastasis of gastric adenocarcinomas. Given the importance of VEGF-A in the pathophysiology of gastric diseases, clarification of its regulation by H. pylori is of pathobiological and clinical relevance.

After establishing vegf-A as host target gene of H. pylori, we characterized the molecular mechanisms underlying vegf-A regulation by the bacterium. Functional analysis of vegf-A 5'-flanking DNA in H. pylori infections identified the sequence -88/-50 as a H. pylori-responsive element of the human vegf-A gene (Fig. 1) . We demonstrate that zinc finger transcription factors Sp1 and Sp3 act as molecular mediators of vegf-A responsiveness to the bacterium and show that H. pylori stimulates Sp1/Sp3 recruitment to the vegf-A -88/-50 element as well as the transactivating capacity of both transcription factors. GC-rich Sp1/Sp3 binding sites within the -88/-50 sequence were found to differentially participate in H. pylori-dependent vegf-A gene regulation. This H. pylori-activated molecular pathway differs from other mechanisms that have been described to control vegf-A expression, as Sp1 and/or Sp3 constitutively bind to the proximal vegf-A promoter in PDGF-treated human fibroblasts and TGF--treated skin keratinocytes without showing changes in their binding patterns upon stimulation. Thus, our findings may allow us to define molecular targets for the modulation of vegf-A gene expression in the setting of H. pylori infection.

We investigated host signal transduction cascades transmitting effects of the bacterium. With kinase phosphorylation analysis and functional transfection studies using dominant-negative kinase mutants, we found that H. pylori stimulates vegf-A transcription through activation of the host MEK>ERK1/-2 kinase module. Although potently activated by the bacterium, MKK4/JNK-dependent signaling cascades do not contribute to the transactivating effect of H. pylori on the vegf-A gene.

In summary, by identifying H. pylori as a potent stimulus of vegf-A gene expression in gastric epithelial cells, our findings further support the concept that the bacterium is able to directly interfere with host angiogenesis and may represent an important aspect of H. pylori pathogenicity. Moreover, our results provide novel molecular insight into mechanisms linking the bacterium to host angiogenesis and so may help to develop innovative strategies to influence vegf-A gene expression in the setting of H. pylori infection.

View larger version (29K): [in this window] [in a new window]   Figure 3. Regulation of host vegf-A expression by H. pylori. H. pylori potently up-regulates release and production of human VEGF-A in gastric epithelial cells; transcriptional activation of the vegf-A gene promoter represents the underlying core mechanism. On the promoter level, enhanced binding of Sp1 and Sp3 to two GC-boxes at -73/-66 and -58/-52 mediates the transactivating effects of H. pylori on the vegf-A gene. Stimulation of Sp1 and Sp3 transactivating capacity represents an additional mechanism of vegf-A regulation by H. pylori. Activation of MEK1>ERK1/2 signaling is crucial for transmission of H. pylori-dependent effects on the human vegf-A gene.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0055fje

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