Role of C-Raf/ MEK1/ ERK2 in Helicobacter pylori-induced gastrin promoter activity

Abstract

Introduction: Helicobacter pylori is a gram negative bacterium, specialized in the colonization of human stomach and a major risk factor for development of gastric cancers. It has been observed in H. pylori-infected patients that gastrin secretion stimulated by H. pylori leads to hypergastrinemia and as consequences, chronic gastritis and gastric atrophy. Gastrin is an important hormone in human stomach which mainly regulates gastric acid secretion. In addition, it is also involved in growth and differentiation of gastric epithelial cells. H. pylori-induced hypergastrinemia is known as a major risk factor for the development of gastric cancers. Moreover, it has been described that H. pylori modulates mitogen activated protein (MAP) kinase pathway, one of the major epithelial cell signaling pathways in humans, which regulates gastrin expression. However, the molecular mechanism, especially how each isotypes of mitogen activated protein kinase pathway are involved in H. pylori-induced gastrin expression, is not fully understood. Purpose: The aim of this study is to explore the molecular mechanism underlying the H. pylori-induced gastrin expression.Materials and methods: The expression level of human gastrin promoter was measured using G240-Luc cells which were constructed by the stable transfection of gastrin promoter-luciferase reporter into AGS cells. In order to identify H. pylori components responsible for the induction of gastrin expression, isogenic mutants of H. pylori G27 wild type; G27ΔcagA, G27ΔcagL and G27ΔPAI were constructed. G27ΔcagA strain lacks cagA gene which encodes the H .pylori major virulence factor CagA, while G27ΔcagL strain lacks cagL gene which is essential for the H. pylori type IV secretion system. G27ΔPAI strain has an entire deletion of cag pathogenicity island encodes the type IV secretion system. After G240-Luc cells were infected by H. pylori G27 or isogenic mutant strains, luciferase activities expressed by the cells were measured and compared. In addition, G240-Luc cells were stimulated with growth factors; Heparin-binding epidermal growth factor like-growth factor (HB-EGF) and Epidermal growth factor (EGF). To knockdown the expression of each signal molecule of MAP kinase pathway, specific siRNAs targeting A-Raf, B-Raf, C-Raf, MEK1, MEK2, ERK1 and ERK2 were used. To verify protein expression knockdown by siRNA treatment, western blot was performed.Results and discussion: G240-Luc cells infected with H. pylori G27 and G27ΔcagA strains showed increased luciferase expression levels and hence increased gastrin promoter activity. In contrast to that, G27ΔcagL and G27ΔPAI H. pylori strains were unable to stimulate the gastrin promoter activity, suggesting that H. pylori type IV secretion system apparatus is important in gastrin promoter activation. In addition, HB-EGF treatment showed a significant gastrin promoter induction which was in similar level to the gastrin promoter induction by EGF treatment, the known agonist of gastrin expression. Both H. pylori-stimulated as well as HB-EGF-stimulated gastrin promoter activities were reduced in the presence of EGFR kinase inhibitor, AG1478. This result suggests that EGFR activation is important in both H. pylori and HB-EGF-induced gastrin promoter activity. As it has been reported that HB-EGF expression was induced by H. pylori, this result leads to speculate that the activation of EGFR via HB-EGF may play a role in H. pylori-induced gastrin expression. C-Raf siRNA treatment showed a significant reduction in H. pylori-stimulated gastrin promoter activity, while A-Raf and B-Raf siRNA treatments didn’t show such reduction in the gastrin promoter activity. This suggests that signal transduction, which lead to gastrin promoter activation by H. pylori is mediated via C-Raf. In addition to that, MEK1-knockdown cells also showed a significant reduction in promoter activity, but knockdown of MEK2 didn’t show any reduction in gastrin promoter activity. This result implies that MEK1 is important in H. pylori-stimulated gastrin expression. This further implies that, MEK1 and MEK2 may play different roles in H. pylori-induced gastrin expression, even though MEK1 and MEK2 proteins function redundantly in general. In the context of ERK, knockdown of ERK2 exhibited significant reduction in gastrin promoter activity, suggesting that ERK2 plays a major role in H. pylori-stimulated gastrin promoter activation. Surprisingly, the knockdown of ERK1 showed a significant induction in H. pylori-induced gastrin promoter activity. This result leads to speculate that ERK1 may exert an inhibitory effect on H. pylori-stimulated gastrin promoter activity, possibly by forming heterodimers withERK2 and ERK2 homodimers may act as potent gastrin promoter activators.Conclusion: H. pylori modulates ERK signal transduction pathway to stimulate gastrin expression via EGFR, C-Raf, MEK1 and ERK2. Moreover, this study reveals for the first time that HB-EGF can stimulate the gastrin promoter activity and thus, EGFR activation via HB-EGF may also play a role in H. pylori-induced gastrin expression. Intriguingly, ERK1 and ERK2 act differently in H. pylori-stimulated gastrin expression. Furthermore, the knowledge of the inhibitory effect of ERK1 in gastrin promoter activation will be helpful to lessen the effect of H. pylori-stimulated gastrin expression. This current understanding of the involvement of MAPK pathway would facilitate the development of therapeutic strategies to reduce H. pylori-induced hypergastrinemia as well as the gastric cancer development.