<i>Helicobacter pylori-</i>induced cell death is counteracted by NF-κB-mediated transcription of DARPP-32.

Abstract

<i>DARPP-32</i> is a frequently amplified and overexpressed gene that promotes several oncogenic functions in gastric cancer. Herein, we investigated the relationship between <i>Helicobacter pylori</i> infection, proinflammatory NF-κB activation and regulation of DARPP-32. The study used <i>in vivo</i> and <i>in vitro</i> experiments. Luciferase reporter, quantitative real-time PCR, immunoblot, chromatin immunoprecipitation (ChIP), cell viability, <i>H. pylori</i> infection, tissue microarrays and immunohistochemical assays were used. Our results indicated that <i>H. pylori</i> infection increased the DARPP-32 mRNA and protein levels in gastric cancer cell lines and gastric mucosa of mice. <i>H. pylori</i> infection increased the activity of NF-κB reporter and p-NF-κB (S536) protein level <i>in vitro</i> and <i>in vivo</i>. To investigate the transcriptional regulation of DARPP-32, we cloned a 3019 bp of the <i>DARPP-32</i> promoter into the luciferase reporter (pGL3-Luc). Both <i>H. pylori</i> infection and tumour necrosis factor-α treatment induced DARPP-32 reporter activity (p<0.01). Using deletion constructs of <i>DARPP-32</i> promoter and ChIP assay, we demonstrated that the sequence -996 to -1008 bp containing putative NF-κB-binding sites is the most active region. The induction of DARPP-32 expression by <i>H. pylori</i> infection counteracted <i>H. pylori</i>-induced cell death through activation of serine/threonine-specific protein kinase (AKT), as determined by ATP-Glo and clonogenic survival assays. Immunohistochemistry analysis demonstrated a significant positive correlation between NF-κB and DARPP-32 expression levels in gastric cancer tissues (r²=0.43, p<0.01). Given the high frequency of DARPP-32 overexpression and its prosurvival oncogenic functions, the induction of DARPP-32 expression following <i>H. pylori</i> infection and activation of NF-κB provides a link between infection, inflammation and gastric tumourigenesis.