CHAC1 overexpression in human gastric parietal cells with Helicobacter pylori infection in the secretory canaliculi

Abstract Background Cation transport regulator 1 (CHAC1), a newly discovered enzyme that degrades glutathione, is induced in Helicobacter pylori (H. pylori)‐infected gastric epithelial cells in culture. The CHAC1‐induced decrease in glutathione leads to an accumulation of reactive oxygen species and somatic mutations in TP53. We evaluated the possible correlation between H. pylori infection and CHAC1 expression in human gastric mucosa. Materials and Methods Both fresh‐frozen and formalin‐fixed paraffin‐embedded tissue samples of gastric mucosa with or without H. pylori infection were obtained from 41 esophageal cancer patients that underwent esophago‐gastrectomy. Fresh samples were used for real‐time polymerase chain reaction for H. pylori DNA and CHAC1 mRNA, and formalin‐fixed samples were used for immunohistochemistry with anti‐CHAC1 and anti‐H. pylori monoclonal antibodies. Double‐enzyme or fluorescence immunohistochemistry and immuno‐electron microscopy were used for further analysis. Results Significant CHAC1 overexpression was detected in H. pylori‐infected parietal cells that expressed the human proton pump/H,K‐ATPase α subunit, whereas a constitutively low level of CHAC1 mRNA expression was observed in the other samples regardless of the H. pylori infection status, reflecting the weak CHAC1 expression detected by immunohistochemistry in the fundic‐gland areas. Immuno‐electron microscopy revealed intact H. pylori cells in the secretory canaliculi of infected parietal cells. Some parietal cells exhibited positive nuclear signals for Ki67 in the neck zone of the gastric fundic‐gland mucosa with H. pylori infection. Conclusion Cation transport regulator 1 overexpression in H. pylori‐infected parietal cells may cause the H. pylori‐induced somatic mutations that contribute to the development of gastric cancer.


| INTRODUC TI ON
Cation transport regulator 1 (CHAC1) is a newly discovered enzyme involved in the γ-glutamyl cycle that can degrade glutathione (GSH) into 5-oxoproline and cysteinyl-glycine. 1,2 CHAC1 is a constituent of the unfolded protein response stress signaling pathway in the endoplasmic reticulum (ER), 3,4 and as a consequence of ER stress, the increase in CHAC1 leads to depletion of GSH and results in unbalanced cellular redox levels. 5 Endoplasmic reticulum stress is triggered by various stimuli, such as infection, inflammation, and gene mutations, 6 and is widely related to the development of several malignant tumors. 7 Helicobacter pylori (H. pylori) infection can mediate ER stress, 8 drastically decreasing GSH levels and increasing the production of reactive oxygen species (ROS) in gastric epithelial cells. [9][10][11] We recently reported that CHAC1 expression is an essential factor driving these sequential changes in infected cells. 12 The decreased GSH and increased ROS lead to somatic mutations in TP53, suggesting that H. pylori-induced CHAC1 overexpression in infected gastric epithelial cells directly contributes to gastric cancer. 12 Helicobacter pylori is a gram-negative, micro-aerophilic spiralshaped bacterium that colonizes the gastric mucosa of the human stomach. More than half of the human population worldwide is infected with H. pylori. 13 Immunohistochemistry (IHC) with a novel anti-H. pylori monoclonal antibody (TMDU-mAb) revealed that H. pylori can be detected not only in the mucus layer attached to the superficial gastric foveolar cells, but also in macrophages scattered in the lamina propria and notably in some parietal cells in H. pylori-infected gastric mucosa. 14 We recently reported that H. pylori infection promotes CHAC1 expression, 12 but the specific gastric cell types where this occurs in vivo in response to H. pylori infection were not clarified. In the present study, we examined gastric mucosa with or without H. pylori infection by real-time reverse transcription polymerase chain reaction (PCR) for CHAC1 mRNA using fresh-frozen tissues and by IHC with a novel anti-CHAC1 monoclonal antibody (CHAC1-mAb (v1v2) ) to locate cells with CHAC1 overexpression in formalin-fixed paraffin-embedded (FFPE) tissue sections.

| RNA extraction and real-time reverse transcription-PCR
To extract RNA, 60-µm-thick fresh-frozen tissue sections were treated with 1.0 mL of TRIzol ® reagent (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized with random primers using Superscript ™ Reverse Transcriptase (Invitrogen). The oligonucleotide primers and probes are listed in Table S2. The relative mRNA quantification was determined by real-time reverse transcription-PCR using the TaqMan Universal PCR Master Mix (ABgene). Amplification and detection were performed with the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems). The amount of target cDNA was normalized with that of the endogenous mRNA of either the housekeeping reference β-actin or the human proton pump/H,K-ATPase α subunit for fresh-frozen stomach tissues.

| Enzyme Immunohistochemistry
Histologic sections (3-µm-thick) cut from FFPE tissue samples were mounted on silane-coated slides (Muto Pure Chemicals Co. Ltd.), de-paraffinized, rehydrated, and pretreated with the appropriate antigen retrieval methods for each antigen. The sections were microwaved for 40 minutes at 97°C to detect CHAC1, H. pylori, and proton pump, or autoclaved for 20 minutes at 121°C to detect the Ki67 antigen, both in 10 mM citrate buffer (pH 6.0). TMDU-mAb and CHAC1-mAb (v1v2) are described elsewhere, respectively. 12,14 The proton pump/H,K-ATPase α subunit, "proton pump," is considered a specific marker of acid-secreting parietal cells. 15 Sections were treated with 3% hydrogen peroxide in methanol for 10 minutes, and then incubated with normal horse serum for Nichirei Bioscience) was used as the chromogen. All specimens were counterstained with Mayer's hematoxylin. Adjacent sections were stained with hematoxylin and eosin staining for further histologic examination.

| Double-enzyme and fluorescence Immunohistochemistry
Histologic sections were processed in the same manner as for enzyme IHC up to the primary antibody reaction. The sections were incubated with alkaline phosphatase-conjugated secondary antibody

| Statistical analysis
Statistical analysis was performed using GraphPad PRISM ver. 6 (GraphPad Software, Inc,). Differences in the CHAC1 mRNA expression between samples with and without H. pylori infection were assessed using the Mann-Whitney U test. A two-tailed P-value of <0.05 was considered statistically significant.

| Cation transport regulator 1 expression in Helicobacter pylori-infected gastric mucosa
Immunohistochemistry with the CHAC1-mAb (v1v2) revealed weak CHAC1 expression, ranging from a few to many cells, in the fundic-gland areas of gastric mucosa with or without H. pylori infection ( Figure 1A). Strong CHAC1 expression in a varying number of cells was observed in the fundic-glands of gastric mucosa with

| Localization of CHAC1 overexpression in Helicobacter pylori-infected gastric mucosa
As CHAC1 seemed to be overexpressed in specific cells of the gastric  Figure 3A and 3). Strong CHAC1 expression was detected in some of these proton pump-positive parietal cells ( Figure 3C and 3). Strong CHAC1 overexpression was also observed in many H. pylori-infected parietal cells (Figure 3E and 3). Taken together, these observations indicated that CHAC1 overexpression was localized in H. pylori-infected parietal cells of the human gastric mucosa.

| Immuno-electron microscopy observation of Helicobacter pylori in parietal cells
Although H. pylori is generally considered a noninvasive extracellular bacterium, many immunoreactive signals were detected inside some parietal cells in many samples with H. pylori infection. Therefore, we used immuno-electron microscopy to further examine the localization of H. pylori in the gastric mucosa ( Figure 4A and B, and Figure 5A and 5). Helicobacter pylori staining in the mucous layer revealed dense rim staining of a whole H. pylori organism, consistent with the assumed distribution of lipopolysaccharides ( Figure 4C). In addition, some bacterial cells that had the same staining patterns were located in the secretory canaliculi of the parietal cells ( Figure 5C).
Higher magnification confirmed that the H. pylori cells embedded in the secretory canaliculi were intact ( Figure 5D and 5).

| Evidence of proliferation found in some parietal lineage cells
Helicobacter pylori infection contributes to the progression of gastric cancer. [16][17][18] Helicobacter pylori infection induces changes to the gastric mucosa and increases cell proliferation, which can eventually lead to inflammation-associated oncogenesis. 19 We examined the extent of parietal cell proliferation in H. pylori-infected samples using double-enzyme IHC with anti-Ki67 antibody to detect proliferation and an anti-proton pump antibody to identify parietal cells. These studies indicated that some parietal cells exhibited positive nuclear signals for Ki67 in the neck zone of the gastric fundic-gland mucosa with H. pylori infection ( Figure 6).

| D ISCUSS I ON
Cation transport regulator 1 is a novel ER stress-inducible gene, and in the presence of ER stress, CHAC1 mRNA levels are upregulated. 3,4 Infection is a factor that stimulates ER stress, 20   Gastric adenocarcinoma is a heterogeneous disease commonly classified into two main histologic types, intestinal and diffuse. 29 Helicobacter infection is a major risk factor for both types. 30 Helicobacter pylori infection is associated with the development of diffuse-type gastric cancer, 31,32 which is thought to develop directly from chronic gastritis without intestinal metaplasia, whereas intestinal-type gastric cancer is thought to develop from atrophic gastritis with intestinal metaplasia. [33][34][35] Gastric cancer arising specifically from parietal cells has not been extensively investigated. Shimada

D I S CLOS U R E O F I NTE R E S T S
No competing interests declared.

AUTH O R CO NTR I B UTI O N S
TO and YW designed the study design, performed most of the experiments, analyzed and interpreted the data, and wrote the manuscript.
PGB contributed to the study design and helped write the manuscript.
KT and TS contributed to the study design. KU, KK, TT, YT, and YN provided the study material and technical support. YE supervised and directed the project, and contributed to the manuscript preparation.