The Role And Mechanism Of PRSS1^R122H And PRSS2 In Hereditary Pancreatitis

Background and Aims:Pancreatitis is a class of inflammatory diseases caused by ectopic overactivation of pancreatic enzymes.Hereditary pancreatitis is a type of autosomal dominant genetic disease,mostly caused by gain-of-function mutants of human PRRS1（cationic trypsinogen）.It eventually progresses to chronic pancreatitis with a significantly increased risk of pancreatic cancer.PRSS1^R122H is the one with the highest carrying rate and the highest penetrance rate in the world,and its risk of pancreatic cancer is about 44%higher than that of the normal population.However,due to the special anatomical location of the pancreas,it is difficult to obtain pancreatic specimens clinically.For a long time,the research on pancreatitis and pancreatic cancer has mainly relied on the use of animal models.However,there is currently no effective treatment for pancreatitis or hereditary pancreatitis.The main reason behind this is the lack of ideal animal models that can truly simulate this type of disease and our deficient understanding of its pathogenesis.However,since the discovery of PRSS1^R122H,the most common mutation in human hereditary pancreatitis 25 years ago,many attempts to develop a genetic mouse model for hereditary pancreatitis by transgenic expression of human PRSS1^R122H have yielded very limited success.Some of these transgenic models can substantially worsen cerulein-indued pancreatitis;however,none of these models showed evidence of spontaneous pancreatitis.For a long time,our understanding of how PRSS1^R122H affects the occurrence and development of pancreatic cancer is quite lacking.In this study,a new humanized transgenic animal model related to hereditary pancreatitis was generated through human bacterial artificial chromosome technology.Based on this novel transgenic mice,we further explored the pathogenesis of hereditary pancreatitis and dissect the role of PRSS1^R122Hin the development of pancreatic tumorigenesis.Methods:1.Use human bacterial artificial chromosome（BAC）as genetic approach to generate PRSS1-PRSS2 transgenic mice.An R122H point mutation(PRSS1^R122H)was introduced by 2 rounds of genetic targeting using Gal K-mediated recombineering.2.PRSS1-PRSS2 and PRSS1^R122H-PRSS2 mice were administrated with a single i.p.injection of cerulein at various doses and pancreatitis was evaluated 24 hours after injection.3.Compare the expression level of PRSS1 and PRSS2 in heterozygous and homozygous state of PRSS1-PRSS2 and PRSS1^R122H-PRSS2 mice with human pancreas lysate.Observe whether there is spontaneous pancreatitis in each of mice.Dissect the role of PRSS2 in the development of spontaneous hereditary pancreatitis.4.Check whether homozygous PRSS1^R122H-PRSS2 mice with advanced chronic pancreatitis has tumorigenesis and the dysregulation of PTEN/Akt pathway.5.Check if there is paternal germline recombination in Ptf1a-Cre male mice.Then,compare the differences in tumorigenesis among the Ptf1a-Cre;PTEN^F/F,PRSS1-PRSS2;Ptf1a-Cre;PTEN^F/F and PRSS1^R122H-PRSS2;Ptf1a-Cre;PTEN^F/F mice and further elucidate its mechanisms.Results:1.Successful generation of BAC transgenic mice containing PRSS1-PRSS2 and PRSS1^R122H-PRSS2 genes.1)BAC RP11-701D14 clone has been verified to contain the PRSS1and PRSS2 gene.2)2 out of 3 Founder Wt BAC transgenic mice carried PRSS1 gene,while 5 out of 7 Founder Mt BAC transgenic mice carried PRSS1 gene.3)Each Founder were further bred with C57BL/6J to generate F1mice.And each of them were verified to contain the CGC>CAC mutation by PCR and Sanger sequencing.2.PRSS1^R122H-PRSS2 mice are more sensitive to cerulein-induced pancreatitis,compared with PRSS1-PRSS2 transgenic mice.1)#2,#3,#4 PRSS1^R122H-PRSS2 mice and#1,#2 PRSS1-PRSS2 mice in F1 generation express both PRSS1 and PRSS2.But#1 PRSS1^R122H-PRSS2 mice only express PRSS1.2)The pancreatitis induced by supramaximal-dose of cerulein in#2,#3 PRSS1^R122H-PRSS2 mice were more severe than#1 and#4.But there is not much difference in cerulein-induced pancreatitis between#1 and#2PRSS1-PRSS2 mice.3)No spontaneous pancreatitis were observed in heterozygous PRSS1-PRSS2 and PRSS1^R122H-PRSS2 transgenic mice at 370 days old.4)After low-dose（2.5μg/kg）cerulein stimulation,only PRSS1^R122H-PRSS2 mice showed obvious edema,relatively high serum amylase level and intra-pancreatic trypsin activity.The pancreatic histology confirmed the typical change of acute pancreatitis.However,when stimulated with supramaximal-dose（100μg/kg）cerulein,the severity of acute pancreatitis in PRSS1-PRSS2 and PRSS1^R122H-PRSS2 mice were similar,and there was no significant difference in the level of trypsin activation between these two mice,both were significantly increased.5)7d after cerulein induction,both of PRSS1-PRSS2 and PRSS1^R122H-PRSS2 mice showed obvious chronic pancreatitis change by macroscopic appearance,pancreas-to-body weight ratio and histology.However,there was no significant difference in the severity of chronic pancreatitis between the two groups.3.Wild-type human PRSS2 and PRSS1^R122H cooperatively initiate spontaneous hereditary pancreatitis in transgenic mice1)Western blot and q PCR showed a high level of PRSS1 and PRSS2expression in the pancreas of PRSS1-PRSS2 and PRSS1^R122H-PRSS2 mice and no expression in WT C57BL/6J mice,compared with human pancreas.Immunohistochemistry staining of PRSS1 and PRSS2 in PRSS1-PRSS2and PRSS1^R122H-PRSS2 mice showed a highly stable and efficient expression of PRSS1 and PRSS2 in acinar cell but not islet and duct cells.After further bred the heterozygous PRSS1-PRSS2 mice into homozygous,western blot showed a similar expression level of PRSS1 and PRSS2 in the pancreas of PRSS1-PRSS2 mice,compared with human pancreas lysate.2)All homozygous PRSS1^R122H-PRSS2 mice but none of the PRSS1-PRSS2 mice,spontaneously developed acute pancreatitis 3 weeks after birth.Homozygous PRSS1^R122H-PRSS2 mice showed obvious early chronic pancreatitis changes around 4 weeks,and adipose tissue replacement began to arise at 6 weeks.The ratio of pancreas weight（mg）to body weight（g）was significantly increased around 3 weeks and decreased significantly at 4 and 6 weeks of age.The increase of serum amylase at about 3 weeks is a further indication of the occurrence of acute pancreatitis.Histopathological changes of the pancreas further confirmed that acute pancreatitis appeared in about 3 weeks,and early chronic pancreatitis began to appear in 4-6 weeks.3)The expression of inflammatory cell markers（CD11b）and macrophage markers（F4-80）increased significantly at 3W,4W and 6W,indicating the massive activation of immune inflammatory cells.4)The expression ofα-SMA in the pancreas from the homozygous PRSS1^R122H-PRSS2 mice at about 4 weeks were significantly increased,indicating the massive activation of pancreatic stellate cells.5)In the 120-day-old homozygous PRSS1^R122H-PRSS2 mice,visible adipose tissue replacement were observed,and 100%of the pancreas appeared to atrophy to varying degrees in all mice,and the ratio of pancreas to body weight was significantly lower than that of the control group.Histopathological changes of the pancreas showed atrophy of acinar cells,fibrosis of pancreatic tissue,fat replacement,and acinar-ductal metaplasia,typical of chronic pancreatitis.Sirius staining indicated that with the development of chronic pancreatitis from early to late stage,a large number of collagen fibers were generated.6)To further dissect the role of PRSS2 in the development of spontaneous pancreatitis,the PRSS1-PRSS2 mice and PRSS1^R122H-PRSS2mice were crossed with another mouse line exclusively expressing human PRSS2 to further increase the level of PRSS2.We observed PRSS2/PRSS1^R122H-PRSS2 compound mice developed spontaneous pancreatitis at 3-weeks-old（Figure 1F）.However,spontaneous pancreatitis was not seen in PRSS2/PRSS1-PRSS2 mice even at 2-months-old.This data strongly supports the idea that wildtype human PRSS2 and mutant PRSS1^R122H can cooperatively initiate spontaneous development of acute pancreatitis.And PRSS2 is not a protective factor in the development of hereditary pancreatitis.4.Pan IN-1 and Pan IN-2 precancerous lesions were observed in the pancreas of 200-day-old homozygous PRSS1^R122H-PRSS2 mice with advanced chronic pancreatitis,accompanied by dysregulation of PTEN/Akt pathway.1)After repeated induction of pancreatitis in homozygous PRSS1-PRSS2 mice,obvious pancreatic fibrosis,chronic inflammation and adipose tissue replacement were observed at 200 days,but no intraepithelial neoplasia occurred.However,Pan IN-1 and Pan IN-2precancerous lesions were observed in the pancreas of 200-day-old homozygous PRSS1^R122H-PRSS2 mice with advanced chronic pancreatitis.2)Western blotting and immunohistochemistry showed that the homozygous PRSS1^R122H-PRSS2 mice around 42d showed significantly higher phosphorylated Akt（locus 473）in pancreatic tissue compared with heterozygous mice.The elevated Akt was due to the significant down-regulation of PTEN expression.3)q PCR showed that the expression of acinar cell marker Amy 2 in homozygous PRSS1^R122H-PRSS2 mice was significantly reduced,while the m RNA levels of ductal cell markers CK19 and SOX9 were significantly increased,suggesting that a large number of acinar cells were transformed into ductal cells.5.PRSS1^R122H in synergy with PTEN inactivation promote the pancreatic tumorigenesis in mice.1)The progenies from male Ptf1a-Cre;PTEN^F/F do not carry the Cre gene,but the pancreas has the phenomenon of suspected PTEN recombination.To examine whether the Ptf1a-Cre will confer paternal recombination,double transgenic mice（hereafter R26/Ptf1a-Cre mice）were generated by crossing Ptf1a-Cre mice with R26（R26-LSL-Lac Z）mice.An R26 locus recombination specific PCR revealed that recombination was present in the pancreas,testes and sperms but not in the tail DNA of the R26/Ptf1a-Cre mice.Positive X-gal staining further supported that recombination occurred not only in the pancreas but also in the germinal center of double transgenic mice.Consistent with these findings,Cre,Lac Z,and Ptf1a m RNA were found in the testes by RT-PCR.R26 locus recombination specific PCR was also detected in the tail DNA of progenies with R26 only.Positive X-gal staining in the lung,liver,and pancreas further confirmed germline recombination transmission.Cre expression occurs before the second meiosis,that is,before the formation of secondary spermatocytes.2)Breeding the PTEN^F/F mice with PRSS1-PRSS2 and PRSS1^R122H-PRSS2 respectively for 2 generations,the possibility of getting homozygous PTEN^F/F;PRSS1-PRSS2 and PTEN^F/F;PRSS1^R122H-PRSS2 is1/16.At the same time,mating the PTEN^F/F with Ptf1a-Cre mice together,there is 1/8 probability of obtaining PTEN^F/F;Ptf1a-Cre mice.Further breeding the homozygous PTEN^F/F;PRSS1-PRSS2 and PTEN^F/F;PRSS1^R122H-PRSS2 with PTEN^F/F;Ptf1a-Cre mice together,the possibility of getting PRSS1-PRSS2;Ptf1a-Cre;PTEN^F/Fand PRSS1^R122H-PRSS2;Ptf1a-Cre;PTEN^F/F is 50%.3)30-day-old Ptf1a-Cre;PTEN^F/F mice（0/20）not only have normal pancreas in general,but also have no changes in pancreatic histopathology.PRSS1-PRSS2;Ptf1a-Cre;PTEN^F/F mice（2/15）had normal pancreas in general,and only 2 of the 15 mice had very limited histological changes in the pancreas.However,Among 30-day-old PRSS1^R122H-PRSS2;Ptf1a-Cre;PTEN^F/F mice,Pan IN-2 lesions were observed in 10/18 mice,Pan IN-3 lesions in 7/18 mice.Of note,one of them have pancreatic cancer histology change.After pancreas-specific PTEN KO,pancreas-to-body weight ratio showed elevated pancreas size in Ptf1a-Cre;PTEN^F/F and PRSS1-PRSS2;Ptf1a-Cre;PTEN^F/F mice.However,PRSS1^R122H-PRSS2;Ptf1a-Cre;PTEN^F/Fmice showed obvious reduced pancreas size due to severe pancreatic leisions.4)After a single injection of high-dose cerulein,PRSS1-PRSS2;PTEN^F/F;Ptf1a-Cre showed a highly elevated intra-pancreatic trypsin activity level,compared with PRSS1-PRSS2;PTEN^F/F mice.5)Western blotting showed that the pancreas-specific knockout of PTEN in PRSS1-PRSS2;Ptf1a-Cre;PTEN^F/F mice have higher expression level of p-Akt（473）,p-Akt（308）than that of PRSS1-PRSS2 without PTEN knockout;The level of total Akt did not change significantly,while the expression levels of PRSS1 and PRSS2 were significantly increased.6)The combined administration of Dabigatran etexilate and Akt inhibitor-MK-2206 can significantly prevent the occurrence and development of pancreatic intraepithelial neoplasia and pancreatic cancer in PRSS1^R122H-PRSS2;PTEN^F/F;Ptf1a-Cre mice.Conclusions:1.A BAC transgene conferred high levels of human PRSS1 and PRSS2 expression in mice,which is the best genetic approach for faithful recapitulation of the human trypsinogens expression.2.Transgenic expression of PRSS1^R122H sensitized mice to cerulein-induced pancreatitis,compared with PRSS1.3.All of homozygous PRSS1^R122H-PRSS2 mice showed spontaneous hereditary pancreatitis.Wild-type human PRSS2 and PRSS1^R122Hcooperatively initiate spontaneous hereditary pancreatitis in transgenic mice4.Homozygous PRSS1^R122H-PRSS2 mice with advanced chronic pancreatitis showed precancerous lesions of Pan IN-1 and Pan IN-2,but not the homozygous PRSS1-PRSS2 mice with recurrent pancreatitis induced by 2 rounds of cerulein stimulation.5.The PTEN/Akt pathway is dysregulated in the homozygous PRSS1^R122H-PRSS2 mice,suggesting that the occurrence of intraepithelial neoplasia may be caused by the inactivation of the PTEN tumor suppressor gene and the activation of the oncogene Akt.6.Pancreas-specific PTEN KO in PRSS1^R122H-PRSS2 mice cause rapid pancreatic tumorigenesis but not in PRSS1-PRSS2 or C57BL/6J mice.A PTEN/Akt pathway-mediated positive feedback loop amplifies Trypsin activity to pathological levels and promote the precancerous lesions and pancreatic cancer formation.7.The trypsin inhibitor dabigatran etexilate combined with the Akt inhibitor MK-2206 treatment can prevent the occurrence of acute pancreatitis-chronic pancreatitis-pancreatic cancer in PRSS1^R122H-PRSS2;Ptf1a-Cre;PTEN^F/F mice.