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. 2017 May;66(5):761-762.
doi: 10.1136/gutjnl-2016-312141. Epub 2016 Sep 2.

Helicobacter pylori- induced cell death is counteracted by NF-κB-mediated transcription of DARPP-32

Shoumin Zhu  1 , Mohammed Soutto  1 , Zheng Chen  1 , DunFa Peng  1 , Judith Romero-Gallo  2 , Uma S Krishna  2 , Abbes Belkhiri  1 , M Kay Washington  3 , Richard Peek  2 , Wael El-Rifai  1   4   5
Affiliations

Helicobacter pylori- induced cell death is counteracted by NF-κB-mediated transcription of DARPP-32

Shoumin Zhu et al. Gut. 2017 May.

Abstract

Objective: DARPP-32 is a frequently amplified and overexpressed gene that promotes several oncogenic functions in gastric cancer. Herein, we investigated the relationship between Helicobacter pylori infection, proinflammatory NF-κB activation and regulation of DARPP-32.

Design: The study used in vivo and in vitro experiments. Luciferase reporter, quantitative real-time PCR, immunoblot, chromatin immunoprecipitation (ChIP), cell viability, H. pylori infection, tissue microarrays and immunohistochemical assays were used.

Results: Our results indicated that H. pylori infection increased the DARPP-32 mRNA and protein levels in gastric cancer cell lines and gastric mucosa of mice. H. pylori infection increased the activity of NF-κB reporter and p-NF-κB (S536) protein level in vitro and in vivo. To investigate the transcriptional regulation of DARPP-32, we cloned a 3019 bp of the DARPP-32 promoter into the luciferase reporter (pGL3-Luc). Both H. pylori infection and tumour necrosis factor-α treatment induced DARPP-32 reporter activity (p<0.01). Using deletion constructs of DARPP-32 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 H. pylori infection counteracted H. pylori-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).

Conclusions: Given the high frequency of DARPP-32 overexpression and its prosurvival oncogenic functions, the induction of DARPP-32 expression following H. pylori infection and activation of NF-κB provides a link between infection, inflammation and gastric tumourigenesis.

Keywords: GASTRIC CANCER; GASTRIC INFLAMMATION; HELICOBACTER PYLORI.

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Figures

Figure 1
Figure 1
Helicobacter pylori infection promotes DARPP-32 expression and activation of NF-κB–P65 in vitro and in vivo. (A) The quantitative real-time (qRT) PCR of DARPP-32 was performed in AGS cells with and without H. pylori infection (7.13 and J166 CagA+ strains). (B) The qRT-PCR analysis of DARPP-32 in gastric tissues collected from mice that were orogastrically challenged with Brucella broth or with CagA+ mouse-adapted H. pylori strain (PMSS1). The expression level of PMSS1 CagA is also shown. (C) Western blot analysis of p-P65 (S536), P65, CagA, cleaved PARP, PARP and DARPP-32 in AGS cells with and without H. pylori infection. (D) Luciferase reporter assay for p-NF-κB-Luc in AGS cells without infection (CTRL), with H. pylori infection alone or in combination with NF-κB inhibition using Bay 11-7082 treatment (BAY). (E) Western blot analysis of p-P65 (S536), P65, CagA, p-AKT (S473), AKT and DARPP-32 using gastric tissues collected from mice that were orogastrically challenged with Brucella broth or with CagA+ mouse-adapted H. pylori strain (PMSS1) for 7 days. MOI, multiplicity of infection.
Figure 2
Figure 2
DARPP-32 does not regulate NF-κB–P65 expression or activation. (A) Western blot analysis of p-P65 (S536), P65 and DARPP-32 proteins in AGS cells stably expressing DARPP-32 (DP32) or empty vector control (pcDNA). (B) p-P65 (S536), P65 and DARPP-32 protein levels were evaluated by immunoblot analysis in control MKN-45/SC shRNA (SC) and MKN-45/DARPP-32 shRNA stable cells (DPshRNA). (C) Luciferase reporter assay for p-NF-κB-Luc in AGS cells stably expressing DARPP-32 or an empty vector control (CTRL) with or without tumour necrosis factor (TNF)-α treatment.
Figure 3
Figure 3
Activation of NF-κB–P65 upregulates DARPP-32 expression. The quantitative real-time (qRT) PCR (A) and western blot (B) analyses of DARPP-32 was performed in AGS cells following tumour necrosis factor (TNF)-α treatment. (C and D) The qRT-PCR and immunoblot analyses of DARPP-32 were performed in MKN-45 cells after Bay 11-7082 treatment. (E and F) The qRT-PCR and immunoblot analyses were performed to determine DARPP-32 expression in AGS cells transiently transfected with P65 or empty vector.
Figure 4
Figure 4
NF-κB–P65 regulates DARPP-32 promoter activity. (A) A scheme showing putative NF-κB transcription factor binding sites on DARPP-32 promoter, and the different deletion constructs of the DARPP-32 promoter (P1–P6). (B) Luciferase reporter assay for various DARPP-32-P (1–6)-Luc constructs in AGS cells expressing P65 or empty vector. (C) Luciferase reporter assay for DARPP-32-P1-Luc, DARPP-32-P2-Luc, DARPP-32-P3-Luc and DARPP-32-P5-Luc in AGS cells treated with tumour necrosis factor (TNF)-α alone or in combination with Bay 11-7082. (D) Luciferase reporter assay for DARPP-32-P5-Luc in AGS cells following transient transfection with empty vector or different amounts of P65. (E) Luciferase reporter assay for DARPP-32-P1-Luc in AGS cells infected with Helicobacter pylori alone or in combination with Bay 11-7082 treatment. (F) Luciferase reporter assay for DARPP-32-P5-Luc in AGS cells infected with H. pylori alone or in combination with Bay 11-7082 treatment.
Figure 5
Figure 5
Chromatin immunoprecipitation (ChIP) assay confirms the binding of NF-κB–P65 on DARPP-32 promoter. (A) ChIP assay by using a specific antibody against P65 to immunoprecipitate formaldehyde-fixed chromatin in AGS cells, followed by regular PCR with primers designed for NF-κB–P65 binding site of DARPP-32 promoter region (P5, as shown in figure 4B). (B) ChIP assay by using a specific antibody against P65 to immunoprecipitate formaldehyde-fixed chromatin in AGS cells, followed by regular PCR with control primers. (C) ChIP assay in AGS cells infected with Helicobacter pylori, followed by regular PCR with primers designed for NF-κB–P65 binding site of DARPP-32 promoter region (P5). (D) ChIP assay in AGS cells infected with H. pylori, followed by quantitative real-time PCR with primers designed for NF-κB–P65 binding site of DARPP-32 promoter region.
Figure 6
Figure 6
Helicobacter pylori activates AKT pathway through regulation of DARPP-32 expression. (A) The clonogenic cell survival assay demonstrates a significant increase in relative colony number in AGS cells stably expressing DARPP-32 (DP32). The data were normalised to uninfected cells; error bars indicate SD. (B) The knockdown of DARPP-32 (DP32) in AGS cells led to a significant reduction in cell viability following H. pylori infection; error bars indicate SD. (C) The clonogenic survival assay shows that the knockdown of DARPP-32 (DP siRNA) in AGS cells led to a significant reduction in number of colonies following H. pylori infection; error bars indicate SD. (D) Western blot analysis of DARPP-32, cleaved PARP, PARP, AKT, p-AKT (S473) and CagA protein following H. pylori infection and transfection with DARPP-32 siRNA (DP siRNA) or control siRNA in AGS cells. (E) The knockdown of DARPP-32 in MKN-45 cells led to a significant reduction in cell survival following H. pylori infection; error bars indicate SD. (F) Western blot analysis of DARPP-32, cleaved PARP, PARP, AKT and p-AKT (S473) proteins following H. pylori infection and transfection with DARPP-32 siRNA (DPsiR) or control siRNA in MKN-45 cells.
Figure 7
Figure 7
Immunohistochemistry for DARPP-32 and p-NF-κB in human gastric tissues. (A and B) Immunohistochemical staining for DARPP-32 and NF-κB in serial tissue sections from human gastric mucosa with normal histology (NG), intestinal metaplasia (IM), high-grade dysplasia (HGD) and adenocarcinoma (AdCa). Original magnification, 20×. A progressive increase in DARPP-32 and p-NF-κB–p65 (S536) coexpression was observed from normal mucosa to adenocarcinoma. The graphs summarise the immunohistochemical staining results on gastric tissue microarrays (C and D). (E) A statistically significant positive correlation between the p-NF-κB (S536) and DARPP-32 composite expression score (CES) was detected (r2=0.434, p<0.01).
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