Trypsin activity governs increased susceptibility to pancreatitis in mice expressing human PRSS1R122H
Fu Gui, Yuebo Zhang, Jianhua Wan, Xianbao Zhan, Yao Yao, Yinghua Li, Ashley N. Haddock, Ji Shi, Jia Guo, Jiaxiang Chen, Xiaohui Zhu, Brandy H. Edenfield, Lu Zhuang, Cheng Hu, Ying Wang, Debabrata Mukhopadhyay, Evette S. Radisky, Lizhi Zhang, Aurelia Lugea, Stephen J. Pandol, Yan Bi, Baoan Ji
Fu Gui, Yuebo Zhang, Jianhua Wan, Xianbao Zhan, Yao Yao, Yinghua Li, Ashley N. Haddock, Ji Shi, Jia Guo, Jiaxiang Chen, Xiaohui Zhu, Brandy H. Edenfield, Lu Zhuang, Cheng Hu, Ying Wang, Debabrata Mukhopadhyay, Evette S. Radisky, Lizhi Zhang, Aurelia Lugea, Stephen J. Pandol, Yan Bi, Baoan Ji
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Research Article Gastroenterology

Trypsin activity governs increased susceptibility to pancreatitis in mice expressing human PRSS1R122H

Abstract

Currently, an effective targeted therapy for pancreatitis is lacking. Hereditary pancreatitis (HP) is a heritable, autosomal-dominant disorder with recurrent acute pancreatitis (AP) progressing to chronic pancreatitis (CP) and a markedly increased risk of pancreatic cancer. In 1996, mutations in PRSS1 were linked to the development of HP. Here, we developed a mouse model by inserting a full-length human PRSS1R122H gene, the most commonly mutated gene in human HP, into mice. Expression of PRSS1R122H protein in the pancreas markedly increased stress signaling pathways and exacerbated AP. After the attack of AP, all PRSS1R122H mice had disease progression to CP, with similar histologic features as those observed in human HP. By comparing PRSS1R122H mice with PRSS1WT mice, as well as enzymatically inactivated Dead-PRSS1R122H mice, we unraveled that increased trypsin activity is the mechanism for R122H mutation to sensitize mice to the development of pancreatitis. We further discovered that trypsin inhibition, in combination with anticoagulation therapy, synergistically prevented progression to CP in PRSS1R122H mice. These animal models help us better understand the complex nature of this disease and provide powerful tools for developing and testing novel therapeutics for human pancreatitis.

Authors

Fu Gui, Yuebo Zhang, Jianhua Wan, Xianbao Zhan, Yao Yao, Yinghua Li, Ashley N. Haddock, Ji Shi, Jia Guo, Jiaxiang Chen, Xiaohui Zhu, Brandy H. Edenfield, Lu Zhuang, Cheng Hu, Ying Wang, Debabrata Mukhopadhyay, Evette S. Radisky, Lizhi Zhang, Aurelia Lugea, Stephen J. Pandol, Yan Bi, Baoan Ji

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Figure 3

Progressive pancreatic damage and activation of stress signaling pathways in PRSS1R122H mice.

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Progressive pancreatic damage and activation of stress signaling pathway...
(A) H&E staining showed progressive pancreatic damage on sections from transgenic PRSS1R122H mice after cerulein-induced AP. In contrast, similarly treated C57BL/6J mice recovered fully (representative of 5 independent samples). Scale bars: 400 μm. (B) Acinar cell death, pancreatic edema, and inflammation evaluation in AP after cerulein induction. Mean ± SEM (n = 5). **P < 0.01; ***P < 0.001; 2-tailed unpaired Student’s t test. (C) Using TUNEL staining, extensive pancreatic acinar cell apoptosis was detected in transgenic PRSS1R122H mice after cerulein induction. In contrast, pancreatic acinar cell apoptosis in C57BL/6J mice was much less prevalent (representative of 5 independent samples). Scale bars: 200 μm. (D) Cleaved caspase 3 was upregulated in the pancreatic acinar cells of PRSS1R122H mice (representative of 5 independent samples). Scale bars: 200 μm. (E) Western blot showed higher levels of p53 and its target gene BAX, DNA damage signaling p-CHK1, and abnormal ER stress-related proteins Bip and CHOP in the pancreata of PRSS1R122H mice than in those from C57BL/6J mice. Representative blots from 3 independent assays are shown. (F) Immunohistochemical staining for 8-hydroxy-2′-deoxyguanosine (8-OH) and phospho-Histone H2A.X (Ser139) (p-H2A.X) indicated the presence of DNA damage in the pancreata of PRSS1R122H mice. Representative of 5 independent samples. Scale bars: 200 μm. (G) mRNA levels of endoplasmic reticulum stress-related signaling molecules Atf4 (activating transcription factor 4) and Xbp1s (X-box binding protein 1 spliced), and ROS-related enzymes Duox1 (dual oxidase 1) and Gpx4 (glutathione peroxidase 4) were measured by real-time RT-PCR. Mean ± SEM (n = 4). *P < 0.05; **P < 0.01; ***P < 0.001; 2-way ANOVA with Tukey’s multiple comparisons test.