Arrestin domain containing 3 promotes Helicobacter pylori–associated gastritis by regulating protease-activated receptor 1
Yu-gang Liu, Yong-sheng Teng, Zhi-guo Shan, Ping Cheng, Chuan-jie Hao, Yi-pin Lv, Fang-yuan Mao, Shi-ming Yang, Weisan Chen, Yong-liang Zhao, Nan You, Quan-ming Zou, Yuan Zhuang
Yu-gang Liu, Yong-sheng Teng, Zhi-guo Shan, Ping Cheng, Chuan-jie Hao, Yi-pin Lv, Fang-yuan Mao, Shi-ming Yang, Weisan Chen, Yong-liang Zhao, Nan You, Quan-ming Zou, Yuan Zhuang
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Research Article Gastroenterology Infectious disease

Arrestin domain containing 3 promotes Helicobacter pylori–associated gastritis by regulating protease-activated receptor 1

Abstract

Arrestin domain containing 3 (ARRDC3) represents a newly discovered α-arrestin involved in obesity, inflammation, and cancer. Here, we demonstrate a proinflammation role of ARRDC3 in Helicobacter pylori–associated gastritis. Increased ARRDC3 was detected in gastric mucosa of patients and mice infected with H. pylori. ARRDC3 in gastric epithelial cells (GECs) was induced by H. pylori, regulated by ERK and PI3K-AKT pathways in a cagA-dependent manner. Human gastric ARRDC3 correlated with the severity of gastritis, and mouse ARRDC3 from non-BM–derived cells promoted gastric inflammation. This inflammation was characterized by the CXCR2-dependent influx of CD45+CD11b+Ly6C–Ly6G+ neutrophils, whose migration was induced via the ARRDC3-dependent production of CXCL2 by GECs. Importantly, gastric inflammation was attenuated in Arrdc3–/– mice but increased in protease-activated receptor 1–/– (Par1–/–) mice. Mechanistically, ARRDC3 in GECs directly interacted with PAR1 and negatively regulated PAR1 via ARRDC3-mediated lysosomal degradation, which abrogated the suppression of CXCL2 production and following neutrophil chemotaxis by PAR1, thereby contributing to the development of H. pylori–associated gastritis. This study identifies a regulatory network involving H. pylori, GECs, ARRDC3, PAR1, and neutrophils, which collectively exert a proinflammatory effect within the gastric microenvironment. Efforts to inhibit this ARRDC3-dependent pathway may provide valuable strategies in treating of H. pylori–associated gastritis.

Authors

Yu-gang Liu, Yong-sheng Teng, Zhi-guo Shan, Ping Cheng, Chuan-jie Hao, Yi-pin Lv, Fang-yuan Mao, Shi-ming Yang, Weisan Chen, Yong-liang Zhao, Nan You, Quan-ming Zou, Yuan Zhuang

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Figure 6

ARRDC3 exerts proinflammatory effects via downregulating PAR1 during H. pylori infection.

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ARRDC3 exerts proinflammatory effects via downregulating PAR1 during H. ...
(A) PAR1 expression in gastric mucosa of uninfected or WT H. pylori–infected WT and Arrdc3–/– mice on day 28 p.i. was analyzed by Western blot. (B) PAR1 expression in gastric mucosa of WT H. pylori–infected BM chimera mice on day 28 p.i. was analyzed by Western blot. (C and D) ARRDC3 siRNA, nonspecific control siRNA (NC), or lipo3000 only (mock) pretreated AGS cells or AGS cells without treatment (medium) were infected with WT H. pylori (MOI = 100) for 24 hours. ARRDC3 and PAR1 expression was analyzed by Western blot (C) or immunofluorescence staining (D). Scale bar: 1 μm. (E and F) ARRDC3 was immunoprecipitated from AGS cells infected with WT H. pylori (MOI = 100, 24 hours) pretreated with or without Bafilomycin A1. PAR1 expression was analyzed by Western blot (E). Interacting PAR1 and precipitated ARRDC3 in the immunocomplexes were further analyzed by Western blotting using anti-ARRDC3 and anti-PAR1. Normal rabbit IgG was used as a negative control (F). (G and H) Histological scores of inflammation (G) and CD45+CD11b+Ly6C–Ly6G+ neutrophil level (H) in gastric mucosa of WT H. pylori–infected WT and Par1–/– mice on day 28 p.i. were compared (n = 6). Representative H&E staining images showed inflammation in gastric antra of WT H. pylori–infected WT and Par1–/– mice on day 28 p.i. Scale bars: 100 μm. (I) PAR1 siRNA, nonspecific control siRNA (NC), or lipo3000 only (mock) pretreated AGS cells or AGS cells without treatment (medium), and primary gastric epithelial cells (GECs) from uninfected WT and Par1–/– mice were infected with WT H. pylori (MOI = 100) for 24 hours. CXCL2 production was measured in cell culture supernatants by ELISA (n = 3). (J and K) Human CD45+CD11b+CD14–CD66b+ neutrophil migration (J) and mouse CD45+CD11b+Ly6C–Ly6G+ neutrophil migration (K) was assessed by transwell assays as described in Methods and statistically analyzed (n = 5). Data are representative of 2 independent experiments. Data are mean ± SEM and analyzed by Student t test, Mann-Whitney U test, and 1-way ANOVA. Western blot results are run in parallel and contemporaneously. *P < 0.05, **P < 0.01, for groups connected by horizontal lines. sup, supernatant.