Regulation of p53 tumor suppressor by Helicobacter pylori in gastric epithelial cells

Abstract

Background & aims: Infection with the gastric mucosal pathogen Helicobacter pylori is the strongest identified risk factor for distal gastric cancer. These bacteria colonize a significant part of the world's population. We investigated the molecular mechanisms of p53 regulation in H pylori-infected cells.

Methods: Mongolian gerbils were challenged with H pylori and their gastric tissues were analyzed by immunohistochemistry and immunoblotting with p53 antibodies. Gastric epithelial cells were co-cultured with H pylori and the regulation of p53 was assessed by real-time polymerase chain reaction, immunoblotting, immunofluorescence, and cell survival assays. Short hairpin RNA and dominant-negative mutants were used to inhibit activities of Human Double Minute 2 (HDM2) and AKT1 proteins.

Results: We found that in addition to previously reported up-regulation of p53, H pylori can also negatively regulate p53 by increasing ubiquitination and proteasomal degradation via activation of the serine/threonine kinase AKT1, which phosphorylates and activates the ubiquitin ligase HDM2. These effects were mediated by the bacterial virulence factor CagA; ectopic expression of CagA in gastric epithelial cells increased phosphorylation of HDM2 along with the ubiquitination and proteasomal degradation of p53. The decrease in p53 levels increased survival of gastric epithelial cells that had sustained DNA damage.

Conclusions: H pylori is able to inhibit the tumor suppressor p53. H pylori activates AKT1, resulting in phosphorylation and activation of HDM2 and subsequent degradation of p53 in gastric epithelial cells. H pylori-induced dysregulation of p53 is a potential mechanism by which the microorganism increases the risk of gastric cancer in infected individuals.

Figure 1. p53 levels are dynamically altered following H. pylori infection in vivo

Upper panel: gastric tissues harvested from gerbils infected with H. pylori strain 7.13 at the indicated time were immunostained for p53 and quantitated using a blind protocol. Results are expressed as the percentage of p53 positive cells per sample. Mean values () for infected and uninfected animals are shown. The blue dashed line connects mean values for p53 expression in infected gerbils. The black dashed line depicts the average levels of p53 in the uninfected control. Lower panel: representative staining for p53 (x20) is shown for uninfected control (1) and infected animals for 4 hrs(2), 8 hrs(3), 24 hrs(4), 3 days(5), 1 week(6), 2 weeks(7), and 12 weeks(8).

Figure 2. Dysregulation of p53 in gastric epithelial cells co-cultured with H. pylori

(A) Protein lysates were prepared from control SNU1 cells (−) or those co-cultured with H. pylori (+) strain J166 for the indicated time and analyzed for expression of p53 by Western blotting (upper panel). The bottom panel shows cells co-cultured with heat-inactivated bacteria. (B) The same as (A) except AGS cells were used. Bottom panel: expression of TAp73β in AGS cells co-cultured with H. pylori. (C) SNU1 cells co-cultured with H. pylori strain J166 or treated with 10 nM camptothecin (CPT) for the indicated time were analyzed for p53 by Western blotting. Expression of p53 protein was quantitated by densitometry and normalized to actin expression. Data depicted as mean ± SEM (n=3). (D) AGS cells treated with 50 nM camptothecin for 6 hours. Camptothecin was removed and cells were then either supplemented with fresh media (upper panel) or co-cultured with H. pylori strain J166 (bottom panel) for 72 hours.

Figure 3. p53 is down-regulated by H. pylori in an HDM2-dependent manner

(A) Left panel: The bar graph represents quantitative real-time RT-PCR analysis of the p53 transcript in AGS cells co-cultured with H. pylori strain J166 for the indicated time. Data were normalized to HPRT1 mRNA. Expression of p53 mRNA in uninfected cells was arbitrarily set at 1. Right panel: Co-culture of AGS cells with H. pylori strain J166 decreases the protein stability of p53 after inhibition of protein synthesis with cycloheximide. Expression of p53 protein was quantitated by densitometry and normalized to actin. Data depicted as mean ± SEM (n=3). (B) Levels of p53 in AGS cells treated with proteasomal inhibitor MG-132 and co-cultured with H. pylori for 24 hours. (C) Treatment of AGS cells with Nutlin3 (10 μM), which interferes with p53-HDM2 interaction, suppresses H. pylori-induced inhibition of p53. (D) Inhibition of HDM2 by siRNA alleviates the down-regulation of p53 induced by H. pylori strain J166 in AGS cells. Right panel: HDM2 siRNA inhibited expression of the HDM2 protein.

Figure 4. AKT protein kinase regulates degradation of p53 in H. pylori-infected cells

(A) Protein lysates were prepared from control AGS and SNU1 cells (−) or those co-cultured with H. pylori strain J166 (+) for the indicated time, and were analyzed by Western blotting with pHDM2 antibody that recognizes phosphorylation at position Ser166. (B) Analysis of MDM2 protein phosphorylation in gerbil gastric tissues after infection with H. pylori strain 7.13 using a pMDM2(Ser166) and MDM2 (154–169) antibodies. (C) Inhibition of AKT by siRNA suppressed H. pylori-mediated degradation of p53 and HDM2 phosphorylation at Ser166 in AGS cells.

Figure 5. CagA regulates p53 levels in H. pylori-infected cells

(A) AGS cells were cultured in the presence of the wild-type H. pylori strain 7.13 or isogenic cagA− or cagE− null mutants, and protein levels of p53 were assessed by Western blotting. (B) Left panel: gastric tissues harvested from gerbils infected with H. pylori strain 7.13 or isogenic cagA-null mutant at indicated time points were immunostained for p53 and quantitated using a blind protocol. Results are expressed as the percentage of p53 positive cells per sample. Mean values () for cagA− and cagA+ infected animals are shown. A dashed line depicts the average levels of p53 in the uninfected control animals. Right panel: representative immunohistochemical staining for p53 (x20) is shown for uninfected animals (1) and those infected with wild-type (2) or cagA− (3) isogenic H. pylori strains for 6 hours (at the peak of p53 increase). Levels of p53 were also analyzed by Western blotting with p53-specific antibody at 6 hours. (C) Analysis of HDM2 phosphorylation in AGS cells co-cultured with H. pylori strain J166 or its isogenic cagA− or cagE− derivatives. (D) Analysis of p53 ubiquitination in AGS cells co-cultured with the indicated isogenic H. pylori strains for 24 hours. Proteasomal degradation was inhibited with MG-132.

Figure 6. CagA induces degradation of p53

(A) Left panel: p53-null Kato III cells were co-transfected with the indicated plasmids and GFP for 48 hours and then analyzed for p53 expression. Gel loading was normalized to GFP expression. HDM2 and p53 co-transfection was used as an additional positive control. Right panel: the same as the left panel but another p53-null osteosarcoma cell line, SaOs2, was used. (B) AGS cells were transfected with CagA-IRES-GFP (CagA) or empty IRES-GFP (Control) vectors. Twenty-four hours post-transfection cells were analyzed by immunofluorescence for p53 (red) in GFP-expressing cells (green). Nuclear p53 protein disappeared in 58% of CagA-expressing cells whereas only 13.5% of control GFP-positive cells were negative for p53. (C) Left panel: AGS cells that express CagA under control of tetracycline-inducible promoter were treated with hydrogen peroxide or left untreated and then analyzed for p53. Right panel: Control AGS cells (−DOX) or ones expressing CagA (+DOX) were treated with indicated concentrations of H₂O₂ for 24 hours. Cell death was assessed by flow cytometry after propidium iodide staining. The proportion of cells in subG1 is shown. CagA significantly increased survival of cells treated with H₂O₂. **, p< 0.01 vs. uninduced cells (n=3). (D) CagA increased long-term survival of AGS cells treated with H₂O₂. Analysis was conducted as described in the Materials and Methods section. The bar graph shows the number of colonies. **, p< 0.01 vs. uninduced cells (n=3).

Figure 7

Regulation of p53 by H. pylori.