ATF6 Regulates The Development Of Chronic Pancreatitis Via Induction Of P53-Mediated Apoptosis

Background and obiectivesChronic pancreatitis(CP)is a progressive,recurrent inflammatory disorder of the pancreas.Initiation and progression of CP can result from serine protease 1(PRSS1)overaccumulation and the ensuing endoplasmic reticulum(ER)stress.However,how ER stress pathways regulate the development and progression of CP remains poorly understood.In the present study we aimed to elucidate the ER stress pathway involved in CP.We found high expression of the ER stress marker genes ATF6,XBP1,and CHOP in human clinical specimens.A humanized PRSS1 transgenic mouse was established and treated with caerulein to mimic the development of CP,as evidenced by pathogenic alterations,collagen deposition,and increased expression of the inflammatory factors IL-6,IL-1β,and TNF-α.ATF6,XBP1,and CHOP expression levels were also increased during CP development in this model.Acinar cell apoptosis was also significantly increased,accompanied by upregulated p53 expression.Inhibition of ATF6 or p53 suppressed the expression of inflammatory factors and progression of CP in the mouse model.Finally,we showed that p53 expression could be regulated by the ATF6/XBP1/CHOP axis to promote the development of CP.We therefore conclude that ATF6 signalling regulates CP progression by modulating pancreatic acinar cell apoptosis,which provides a target for ER stress-based diagnosis and treatment of CP.Experimental materials1.Human pancreas tissue specimenPancreatic tissues were collected from 23 CP patients(mean age=44.57±19.79)and 31 patients with benign pancreatic tumours or peritumoural normal pancreatic tissues(mean age=43.13 ± 13.93)as controls.All patients were registered at the Department of Hepatobiliary Surgery in Nanfang Hospital,Southern Medical University in Guangzhou,China between 2014 and 2018.The research procedures were approved by the Ethics Committee of Southern Medical University,and written informed consent was obtained from each patient before the study.2.Experimental animalsHealthy male C57BL/6 mice aged 5-6 weeks were used in this study.All mice were maintained in standard experimental cages at 24 ± 2℃ under a 12 h light/dark cycle and supplied with standard laboratory animal chow and water ad libitum.The experimental operations were performed with approval from the Institutional Animal Care and Use Committee of Southern Medical University.Experimental methods1.Establishment of PRSS1 transgenic mice,ATF6 knock out mice and ATF6 adenovirusHumanized PRSS1 transgenic mice and ATF6 knock mice were constructed.ATF6 adenovirus including ATF6 overexpression,shATF6 for ATF6 inhibition and negative control were also constructed for functional research.2.CP induction and treatmentTo establish a CP animal model,male transgenic mice were intraperitoneally injected with 15 μg/mL caerulein dissolved in phosphate-buffered saline at 50 μg/kg each hour for 8 h.3.IHC and immunofluorescence assaysH&E staining and IHC analysis of pancreatic tissue slides for ATF6,CHOP,XBP-1,collagen I,a-SMA,and p53 were performed according to a previously described protocol[40].4.Masson’s trichrome stainingThe collagen fibre content of pancreatic tissues was determined using a Masson’s Trichrome Stain Kit(G1340;Solarbio Science&Technology,Beijing,China)according to the manufacturer’s instructions.5.Quantitative RT-PCRQuantitative RT-PCR was performed in this study to detect gene mRNA levels in primary acinar cells or pancreatic tissues.6.Western blottingTotal protein was extracted from the mouse pancreatic tissues.Primary antibodies used in this study include antiATF6,anti-CHOP,anti-XBP-1,anti-p53,and anti-GAPDH.The protein abundance was evaluated by immunoblotting with at least three biological replicates.7.TUNEL assayCell apoptosis in mouse pancreatic tissues was analyzed using the TUNEL method with a DNA Fragmentation Imaging Kit(Sigma Aldrich)as instructed by the manufacture.8.Statistical analysisStatistical analysis was carried out using GraphPad Prism 8.3 and SPSS 20.0 software,and data are presented as the mean± the standard error of the mean.Significant differences between two groups were analyzed by Student’s t-test,and one-way analysis of variance was performed to investigate the differences among more than two groups.Significant differences were defined by a P value of<0.05.Results1.CP patients have enhanced ER stress responsesWe collected pancreatic tissues from CP patients and healthy volunteers.Pancreatic tissues from CP patients showed histological and cellular alterations typical of CP:increased collagen in the peri-acinar areas,frequent disappearance and vacuolization of acinar cells,and substantial pancreatic impairment.Using transmission electron microscopy,we observed that the ER was broken into fragments and bubbles of different sizes in human CP tissues,which indicated ER stress activation.Subsequently,we carried out an immunohistochemistry(IHC)analysis of effector proteins in the ER stress pathways.Compared with those in healthy volunteers,the protein levels of activating transcription factor 6(ATF6),C/EBP-homologous protein(CHOP),and X-box-binding protein 1(XBP1)were significantly increased in pancreatic tissues from patients with CP.2.ER stress responses are enhanced during CP progression in PRSS1 transgenic micewe established a mouse CP model by treating humanized PRSS1 transgenic mice with caerulein.More severe CP manifestation observed in PRSS1 transgenic mice than in wildtype mice confirmed PRSS1 overexpression as a driving force for the pathogenesis of CP.Next,we observed a gradual increase in ER destruction in the pancreatic tissues in the mouse CP model,indicating an increased activation of the ER stress response.3.Acinar cell apoptosis and p53 levels are increased in the mouse CP modelA transferase-mediated d-UTP nick-end-labelling(TUNEL)assay showed that numbers of apoptotic cells in PRSS1 mice pancreatic tissues were remarkably increased 1 and 2 weeks after caerulein treatment.we measured levels of p53,a master regulator of apoptosis,in the CP model.Caerulein treatment significantly induced p53 mRNA and protein levels in pancreatic tissues from PRSS1 transgenic,with the highest levels observed 1 and 2 weeks after treatment,respectively.4.ER stress regulates p53 during CP progressionwe inhibited p53 expression and the ER stress response in the mouse CP model using pifithrin-α(PFT-α)and tauroursodeoxycholic acid(TUDCA),respectively.We observed that p53 expression was significantly suppressed by both PFT-α and TUDCA treatments,indicating that ER stress could effectively regulate p53 expression during CP pathogenesis.5.ATF6 promotes p53 expression,inflammation,and acinar cell apoptosis in vitroprimary acinar cells were isolated from PRSS1 transgenic mice and treated with lipopolysaccharide(LPS).We observed that ATF6 and p53 expression was induced by LPS in primary acinar cells.The increase in p53 expression in primary acinar cells induced by LPS treatment was greatly suppressed by ATF6-interfering adenovirus(shATF6).Additionally,inhibition of ATF6 expression resulted in significant decreases in IL-6 and IL-1β levels,but not in TNF-α,in cell culture media from LPS-treated primary acinar cells.The apoptosis of primary acinar cells,which was promoted by LPS treatment,was also suppressed by ATF6 inhibition.Moreover,LPS induced p53 expression in acinar cells from PRSS1 transgenic mice was substantially decreased by ATF6-interfering adenovirus.These in vitro results show that ATF6 promotes p53 expression,inflammation,and acinar cell apoptosis.6.ATF6 promotes p53 expression and inflammation in vivoFor further confirmation of the effect of ATF6 on p53 expression and CP pathogenesis,PRSS1 transgenic mice were crossed with ATF6 knockout mice.Four weeks after caerulein treatment,p53 expression in PRSS1 transgenic ATF6 knockout mice was lower than that in wild-type mice and PRSS1 transgenic mice treated with caerulein.In contrast,ATF6 gene overexpression increased p53 expression in PRSS1 transgenic ATF6-/-mice 4 weeks after caerulein treatment.In addition,we observed that levels of the inflammatory factors IL-6,IL-1β,and TNF-α in the pancreatic tissues of PRSS1 transgenic mice after CP induction by caerulein were significantly reduced by ATF6 gene knockout but rescued by ATF6 overexpression to levels comparable with those in PRSS1 transgenic mice after CP induction.ConclusionATF6 in ER stress pathway affects the progress of CP by regulating p53-mediated apoptosis.