Helicobacter pylori induction of the gastrin promoter through GC-rich DNA elements

Abstract

Background: Helicobacter pylori (H. pylori) infection has been linked to the development of chronic gastritis, duodenal ulcer disease, and gastric cancer. Helicobacter pylori- infected patients and animal models develop hypergastrinemia, chronic gastritis, and gastric atrophy. Since gastrin is an important regulator of gastric acid secretion and cell growth, H. pylori regulation of this hormone has been implicated in its pathogenesis.

Objectives: To investigate the effect of H. pylori on gastrin gene expression in mice and of human bacterial isolates on gastrin mRNA expressed in a human cell line.

Methods: Gastrin mRNA was measured by qRT-PCR in H. pylori-infected mice. H. pylori were co-cultured with AGS cells to study regulation of human gastrin gene expression. Various MAP kinases were implicated in signal transduction from the bacteria using specific inhibitors. Gastrin reporter constructs and gel shift assays were used to map DNA responsive elements.

Results: In addition to an increase in gastrin mRNA in H. pylori-infected mice, H. pylori induced the endogenous human gastrin gene through MAP kinase-dependent signaling but not NFκB-dependent signaling. Activation of gastrin through MAPK signaling did not require CagA or VacA virulence factors. Transfection studies demonstrated that a GC-rich motif mediated H. pylori-induction of the gastrin promoter and that the motif inducibly binds Sp1 and Sp3 transcription factors.

Conclusions: Direct contact of live H. pylori bacteria with human cells is sufficient to induce gastrin gene expression.

Figure 1. H. pylori Stimulate Gastrin Gene Expression in Infected Mice

Mice were infected with H. pylori SS1and analyzed six months after infection. qRT-PCR analysis of gastrin normalized to GAPDH (Gastrin mRNA) was performed using RNA isolated from the stomachs of infected and uninfected (—) mice (N = 5 mice). The mean is indicated by a bar. *P < 0.05.

Figure 2. H. pylori Stimulate Expression of Gastrin in AGS Cells

AGS cells were infected with H. pylori strain 26695 (a human isolate) 48h after serum starvation. The cells were cultured with bacteria at a m.o.i of 100 to 1 at various times (A) or with increasing amounts of bacteria (m.o.i) for 9h (B). All subsequent H. pylori co-culture experiments were performed at 100 to 1 m.o.i. for 9h. AGS cells were co-cultured with live or inactive (formalin fixed, heat-treated) bacteria (C). qRT-PCR analyses of gastrin and 18S rRNA transcripts were performed on total RNA and the amount of gastrin mRNA was normalized to 18S rRNA (Gastrin mRNA). All samples were compared to the untreated sample which was set to 1. The mean fold change + SEM is shown for three independent experiments. *P < 0.05 compared to untreated cells was considered significant. (D) AGS cells were co-cultured with the wild type H. pylori strain 26695 (WT), or isotypes mutants of 26695 that were null for CagA (-CagA) or VacA (-VacA). Two additional human Hp isolates were also used, the wild type J99 strain and the mouse-adapted SS1 strain. qRT-PCR analyses of gastrin and 18S rRNA transcripts were performed. Gastrin mRNA was normalized to the 18S transcript. The mean fold change + SEM three experiments is shown. *P< 0.05 compared to untreated cells was considered significant.

Figure 3. MAP Kinase Inhibitors Block H. pylori -induced Gastrin Expression

AGS cells were treated with vehicle alone (—) or with chemical inhibitors of ERK, JNK and p38 MAP Kinases respectively (PD98059: 50μM, SP600125: 10μM, SB203580: 10μM) or NFκB (APDT: 10μM) signaling 30 min prior to co-culture with the H. pylori 26695 strain (Hp). qRT-PCR analysis of gastrin transcripts (A) or IL-8 transcripts (B) were normalized to 18S rRNA. The mean fold change + SEM three experiments is shown *P< 0.05 compared to untreated (—) cells. Western blots were performed on whole cell extracts prepared from cells cultured without (—) or with (+) H. pylori ± the indicated inhibitors (C). (D) AGS cells were treated without (—) or with 1μM α-amanitin prior to co-culture with the H. pylori 26695 strain. Gastrin mRNA normalized to 18S rRNA transcripts was determined by qRT-PCR of total RNA. The mean fold change + SEM for three experiments is shown. *P < 0.05 compared to untreated cells.

Figure 4. H. pylori Stimulate the Proximal Gastrin Promoter

AGS cells were transiently transfected with the empty vector (pGL3), a series of gastrin luciferase constructs, or an AP1 luciferase reporter (pAP1-Luc). A schematic representation of the gastrin luciferase constructs is shown (A). The basal activity of the gastrin constructs is shown as relative light units (RLU) of the firefly normalized to the Renilla luciferase activity (B). Cells were co-cultured with H. pylori (C) or 100 nM PMA (D) 48h post-transfection. The mean fold change + SEM for three separate experiments was performed in triplicate. The filled bar represents the baseline expression in RLU for each construct set to 1 and fold changes were the ratio of the stimulated activity compared to basal expression of each construct.

Figure 5. H. pylori Stimulate the Gastrin Promoter Through GC-rich DNA Elements

AGS cells were transiently transfected with the wild type (WT) 240 bp gastrin luciferase reporter or the reporter containing point mutations at the indicated sites (S1,2,3). Altered nucleotides are shown in bold font (A). The basal activity of gastrin constructs is shown as relative light units (RLU) of firefly to Renilla activity (B). The cells were co-cultured with H. pylori 48 h post-transfection (C). The mean fold change + SEM for three separate experiments was performed in triplicate. The filled bar represents the baseline expression in RLU for each construct set to 1 and fold changes were the ratio of the stimulated activity compared to basal expression.

Figure 5. H. pylori Stimulate the Gastrin Promoter Through GC-rich DNA Elements

AGS cells were transiently transfected with the wild type (WT) 240 bp gastrin luciferase reporter or the reporter containing point mutations at the indicated sites (S1,2,3). Altered nucleotides are shown in bold font (A). The basal activity of gastrin constructs is shown as relative light units (RLU) of firefly to Renilla activity (B). The cells were co-cultured with H. pylori 48 h post-transfection (C). The mean fold change + SEM for three separate experiments was performed in triplicate. The filled bar represents the baseline expression in RLU for each construct set to 1 and fold changes were the ratio of the stimulated activity compared to basal expression.

Figure 6. H. pylori Stimulate Binding of Sp1 and Sp3 to the GC-rich DNA Elements

EMSA reactions were carried out with 4μg of nuclear extracts from AGS cells that were untreated (—) or co-cultured with H. pylori 26695 strain for 3 h (Hp). Labeled probes represent GC-rich DNA elements of the human gastrin promoter and a consensus AP1 binding sequence. The samples were incubated with antibodies against Sp3 or Sp1 as indicated. Arrows indicate the supershifted Sp1 and Sp3 DNA complexes.