The Role And Mechanism Of Trypsinogen In Chronic Pancreatitis-oncogenic Transformation

Part 1.Pathogenetic roles of trypsinogen mutation in chronic pancreatitis of miceObjective: Mutations in the trypsinogen gene（PRSS1）cause human hereditary chronic pancreatitis.However,it is not clear how mutant forms of PRSS1 contribute to disease development.We studied the effects of expressing mutant forms of human PRSS1 in mice.Methods: We expressed forms of PRSS1 with and without the mutation encoding R122H(PRSS1^R122H)specifically in pancreatic acinar cells under control of a full-length pancreatic elastase gene promoter.Mice that did not express these transgenes were used as controls.Mice were given intraperitoneal injection of lipopolysaccharide（LPS）to induce chronic pancreatitis.Other groups of mice were fed ethanol or placed on a high-fat diet to induce chronic pancreatitis.Pancreata were collected and analyzed by histology,immunoblots,real-time polymerase chain reaction,and immunohistochemistry.Trypsin enzymatic activity and chymotrypsin C（CTRC）enzymatic activity were measured in pancreatic homogenates.Results: Pancreata from mice expressing transgenes encoding PRSS1 or PRSS1R122 H had focal areas of inflammation;these lesions were more prominent in mice expressing PRSS1R122 H.Compared with control mice,pancreata from mice that express PRSS1 or PRSS1R122 H had an increased level of NRF2 and reduced level of CTRC（adaptive responses）,and no difference in trypsin activity,which did not lead to spontaneous chronic pancreatitis.Administration of lipopolysaccharide exacerbated inflammation in mice that express PRSS1R122 H compared to mice that express PRSS1 or control mice.Mice that express PRSS1R122 H developed more severe chronic pancreatitis after ethanol feeding or a high-fat diet than mice that express PRSS1 or control mice.Pancreata from mice that express PRSS1R122 H had more DNA damage,apoptosis,and collagen deposition and increased trypsin activity and infiltration by inflammatory cells than mice that express PRSS1 or control mice.Damage of PRSS1R122 H combined with environmental insults can break the homeostasis caused by protective responses and cause severe chronic pancreatitis.Conclusions: Expression of a transgene encoding PRSS1R122 H in mice promoted inflammation and increased the severity of chronic pancreatitis compared with mice that express PRSS1 or control mice.These mice might be used as a model for human hereditary pancreatitis and can be studied to determine mechanisms of induction of chronic pancreatitis by lipopolysaccharide,ethanol,or a high-fat diet.Part 2.The influence of PRSS1R122 H mutation on chronic pancreatitis induced by environmental insults combined with Kras mutationObjective: To determine whether chronic inflammation caused by trypsinogen mutation interacts with endogenous Kras mutation in mouse chronic pancreatitis under the influence of environmental insults.Methods: Tool mice BAC Elastase-Cre ERT were hybridized with Loxp-STOP-Loxp driven expression of endogenous KrasG12D/+ mutation mice（LSL-KrasG12D/+,LSL）to generate BAC Ela Cre ERT;Kras G12D/+ double transgenic mice（LSL/BAC）,which can induce Cre recombinase activity and acinar specific Loxp-STOP-Loxp splicing by Tamoxifen administration to specifically express endogenous KrasG12 D mutation in all pancreatic acinar cells.The LSL/BAC mice were then hybridized with the transgenic mice specifically expressing human PRSS1 gene（PRSS1）and encoding PRSS1R122 H mutation（R122H）in pancreatic acinar cells respectively as described in the part one.Triple transgenic mice of PRSS1/LSL-KrasG12D/BAC（P/L/B）and PRSS1^R122H/LSL-KrasG12D/BAC（R/L/B）were obtained,and specific expression in mouse pancreatic acinar cells of human PRSS1 and single mutation of KrasG12 D,amphimutation of PRSS1R122 H and KrasG12 D were induced by Tamoxifen,respectively.Genotyping was performed to confirm the genotypes.The mice were respectively fed with high fat diet or alcohol diet for 28 days,and then sacrificed.Pancreaitic tissues were harvested for pathological evaluation,sirius red staining,detection of fibrosis markers,immunohistochemical staining for inflammatory cell markers,TUNEL apoptosis detection and immunofluorescence detection of cleaved caspase 3,trypsin activity and CTRC activity assay.Results: Compared with P/L/B mice,H&E staining of pancreatic tissues from R/L/B mice fed with high fat or alcohol diet showed more severe chronic pancreatitis,characterized by more acinar cell atrophy,inflammatory cells infiltration and fibrosis.Sirius red staining showed that R/L/B mice had more collagen deposition in pancreas than P/L/B mice,and the m RNA and protein expression levels of collagen-1（fibrotic marker）and α-SMA（pancreatic stellate cell activation marker）in R/L/B group were significantly higher than those in P/L/B group.The positive immunohistochemical staining areas of inflammatory cell markers（F4/80,CD45 and MPO）in R/L/B group were significantly larger than those in P/L/B group.TUNEL signals representing the level of apoptosis and immunofluorescence positive cells of cleaved caspase 3 in R/L/B group were significantly higher than those in P/L/B group.The trypsin activity of mice in R/L/B group was significantly higher than that in P/L/B group,but there was no significant difference in trypsin activity between R/L/B group and R/B group mice.The m RNA and protein expressions of CTRC in mice with gene mutation were lower than those in BAC control mice.There was no significant difference in CTRC expression between the R/L/B mice with amphimutation and the R/B mice with only R122 H mutation.Conclusions: PRSS1R122 H mutation can aggravate chronic pancreatitis caused by environmental insults combined with KrasG12 D mutation,and PRSS1R122 H mutation was the initiating factor.The mechanism was that the amphimutation can increase trypsin activity,cause acinar cell apoptosis,and exacerbate inflammation.Part 3.Synergistic effect and mechanism of PRSS1R122 H combined with Kras mutation in chronic pancreatitis-oncogenic transformationObjective: To investigate whether the amphimutation of PRSS1R122 H and KrasG12 D play a synergistic role in chronic pancreatitis-oncogenic transformation,and to explore the possible mechanism.Methods: Using BAC control mice,PRSS1/LSL-KrasG12D/BAC（P/L/B）and PRSS1^R122H/LSL-KrasG12D/BAC（R/L/B）triple transgenic mice as models,we observed the differences of oncogenic phenotypes and changes of related pathways,which were verified by in vivo and in vitro experiments.After 28 days’ treatments of high fat or alcohol diet,mice in the three groups were sacrificed for pancreatic tissues harvest.H&E staining was performed to observe the histological changes of ADM and Pan IN,and the areas were counted.The commonly used markers of ADM and Pan IN were detected by immunohistochemical staining and immunofluorescence.“Pulldown” assay by agarose beads was used to detect Kras activity;western blot was used to detect protein expression of downstream ERK and AKT/m TOR pathways;and q RT-PCR,western blot and immunohistochemical staining were used to detect expression of ATF3 and NF-κB.The primary acinar cells isolated from transgenic expressed BAC,P/L/B and R/L/B mice were3 D cultured.Alcohol was added to stimulate the acinar cells to observe whether there was transdifferentiation to ductal cells.Results: After a high fat and alcohol diet feeding,R/L/B mice with amphimutation developed more severe ADM and Pan IN.Immunohistochemical staining and immunofluorescence were used to detect the changes of ADM markers（Amylase,MIST 1and CK19）.Immunohistochemical staining detecting Muc5 and Ki-67 and Alcian blue staining were used to determine the range of Pan IN.All results demonstrated that changes were more obvious in the pancreas of R/L/B mice.Kras activation assay showed that the content of activated Kras protein in pancreas of R/L/B mice was higher than that in P/L/B mice,and the expression of downstream p ERK protein,ATF3 and NF-κB were also higher in R/L/B mice.The 3D cultured acinar cells from R/L/B mice were stimulated by alcohol and formed more ductal structures.Conclusions: PRSS1R122 H mutation combined with environmental insults can lead to more severe endoplasmic reticulum stress in acinar cells,and ATF3 expression was significantly up-regulated,which activated the NF-κB pathway,and positive feedback continuously activated Kras activity and downstream ERK pathway,causing the occurrence of ADM and Pan IN.Summing up the above research,we can come to the conclusions as follows:Trypsinogen PRSS1R122 H mutation can aggravate environmentally induced chronic pancreatitis,resulting in more severe endoplasmic reticulum stress in acinar cells,significantly upregulated ATF3 expression,and activation of NF-κB pathway,which triggered a “second hit” to mice with Kras G12 D mutation,sustained activation of Kras and downstream ERK pathway.Ultimately acinar apoptosis,ADM and Pan IN were induced.PRSS1R122 H can aggravate KrasG12 D mutation-mediated chronic pancreatitis-oncogenic transformation induced by environmental insults.This study for the first time links the interaction of genetic mutations and environmental factors in chronic pancreatitis-oncogenic transformation and validates the association in model animals,contributing to in-depth understanding of the mechanism of chronic pancreatitis-oncogenic transformation,providing reference for the clinical management of chronic pancreatitis patients with amphimutation,and potential targets for better prevention and treatment of this pathological process.