The Role Of FAT10 Regulation Of HIF1α Expression In The Pathogenesis Of Acute Pancreatitis In Mice

Background and Objective: Acute pancreatitis(AP)is one of the most frequent acute abdomen in clinic.Its incidence rate is high in recent years.The onset of the disease is often sudden,which is a complex pathological process involving a variety of pathogenic factors,so the symptoms of the patients are often different.Mild acute pancreatitis and severe acute pancreatitis can be classified according to the condition.Mild acute pancreatitis accounts for about 80%,often mild,self limiting development,low mortality,good prognosis.Severe acute pancreatitis(SAP)is often accompanied by obvious systemic inflammatory response syndrome,showing serious clinical symptoms.At present,there is no good specific plan for the treatment of this disease except for symptomatic and supportive treatment.Therefore,the fatality rate of the disease is relatively high,reaching about 30%,and its high treatment costs also make it difficult for most patients to afford it.However,the pathogenesis of acute pancreatitis is still unclear,so it is the most important to explore the pathogenesis and treatment of acute pancreatitis.FAT10(human HLA-F adjacent transcript 10)is a member of ubiquitin like superfamily.It has the function of degrading substrate protein similar to ubiquitin(UB),and participates in the process of protein post translational modification(PTM).FAT10 has potential functions in regulating cell cycle,tumorigenesis,inhibiting cell proliferation and survival.The increased expression of FAT10 was also found in a variety of cancers,suggesting that FAT10 is involved in the tumor process.Our previous studies also showed that FAT10 can affect the occurrence and development of liver cancer.In recent years,it has been suggested that FAT10 may play an important role in the regulation of inflammatory response,but the specific mechanism is unknown.Hypoxia inducible factor(HIF)is a major regulatory factor of cells under hypoxia.Under hypoxia relief,cells can activate hypoxia-related signaling pathways and enhance their tolerance to hypoxia.As an important hypoxia regulatory protein,HIF1α plays an important role in inflammation.It has been reported earlier that HIF1α knockout affects macrophage infiltration into tissues and plays an important role in the recruitment of inflammatory cells.Recent studies have shown that HIF1α plays a dual role in pancreatic exocrine homeostasis and in the progression of pancreatic coagulation-induced acute pancreatitis.However,the relationship between FAT10 and HIF1α is not clear and the specific role of FAT10 in the pathogenesis of acute pancreatitis has not been reported.Therefore,this study intends to establish a mouse model of acute pancreatitis to clarify the specific role of FAT10 and explore its specific pathogenesis.Methods: 1.A mouse model of acute pancreatitis was established,and the expressions of amylase and lipase in serum of wild-type mice and FAT10 knockout mice were determined by biochemical analysis.Then,in order to clarify the effect of FAT10 on the severity of inflammation,mice were sacrificed and pancreatic tissues were taken to detect the activities of trypsin and myeloperoxidase in the pancreatic tissues,and pancreatic edema was observed.HE staining and immunohistochemistry were used to observe the severity of inflammation and macrophage infiltration in the pancreatic tissue of mice.2.The role of FAT10 in macrophage recruitment was determined by Transwell experiment in vitro.Next,FAT10 plasmid was transfected with interference and overexpression in mouse acinus cancer cells(266-6).The changes of HIF1α protein and the recruitment of macrophages were detected by WB assay and Transwell assay.Furthermore,we constructed the HIF1α knockout 266-6 cell line,and used Transwell assay to observe whether the recruitment of macrophages by FAT10 depends on HIF1α.3.WB assay and Quantitative Real-time PCR were used to detect the changes of VHL protein and m RNA under the condition of changing FAT10 by various methods.Then,WB assay was used to detect the relationship between FAT10 and HIF1α ubiquitination levels in HEK293 T cells and 266-6 cells.Further,WB assay was conducted to observe the changes of VHL,HIF1α protein and ubiquitination levels in the condition of changing FAT10.4.The WB experiment confirmed that FAT10 regulation of VHL expression was independent of UB.Then,WB experiment was conducted to observe whether changing FAT10 could regulate the proteasomal degradation of VHL.Furthermore,IP experiment and confocal microscope were used to observe whether FAT10 and VHL had a binding relationship,and at the same time,to determine whether FAT10 could make VHL FAT10 ylation.Finally,HIF1α ubiquitination was observed in FAT10 knockout 266-6 cells and HEK293 T cells by TNF-α stimulation or FAT10 overexpression plasmid transfection.5.The combination of FAT10 and HIF1α was observed through IP experiment.Subsequently,the expression of FAT10 was altered in A498 cells with natural deletion of VHL gene to determine the change of HIF1α ubiquitination and FAT10-HIF1α complex level.Further,WB and IP experiments were performed to observe whether FAT10-HIF1α complex could be degraded by proteasome and to clarify the relationship between FAT10-HIF1α complex and HIF1α protein expression.Results: 1.A mouse model of acute pancreatitis was established.Firstly,we confirmed the high expression of FAT10 in the pancreatic tissue of mice with acute pancreatitis through a variety of experimental methods.Serum biochemical analysis of wild-type mice and FAT10 knockout mice showed that the levels of serum amylase and lipase increased rapidly in wild-type mice.Interestingly,however,serum amylase and serum lipase levels were also increased in FAT10 knockout mice,but the increase was not significantly greater than in wild-type mice.By detecting the activities of trypsin and myeloperoxidase in pancreatic tissue,it was found that compared with wild-type mice,the increase of trypsin and myeloperoxidase activities in pancreatic tissue of FAT10 knockout mice was not significantly higher than that of wild-type mice.By measuring the water content in the pancreatic tissue of mice,the degree of pancreatic tissue edema of FAT10 knockout mice was significantly reduced compared with that of wild-type mice.HE staining further revealed that wild-type mice showed obvious pancreatic tissue inflammation after being stimulated by cerulein.However,although the pancreatic tissue of FAT10 knockout mice also showed inflammation,it was significantly milder than that of wild-type mice,and the pathological index was significantly lower.Finally,immunohistochemical experiments showed that macrophage infiltration was significantly reduced in the pancreatic tissue of FAT10 knockout mice.2.Transwell experiments demonstrated that TNF-α stimulation significantly increased macrophage recruitment in wild-type mouse pancreatic primary acinus cells and mouse acinus cancer cells(266-6)in vitro.However,TNF-α stimulation did not significantly increase macrophage recruitment in primary pancreatic acinus cells of FAT10 knockout mice and FAT10 knockout 266-6 cells.Subsequently,FAT10 interference and overexpression plasmid were transfected in 266-6 cells.WB and Transwell experiments showed that the expression of HIF1α decreased after FAT10 interference,and the expression of the downstream VEGF protein of HIF1α also decreased,and the recruitment of macrophages decreased.However,after FAT10 upregulation,the expression of HIF1α increased,and the expression of VEGF protein also increased,and the recruitment of macrophages increased significantly.Finally,we altered the expression of FAT10 in HIF1α knockout 266-6 cells.WB results showed that in 266-6 cells with HIF1α knockout,FAT10 expression was changed,HIF1α protein was not expressed,and VEGF protein expression was not significantly changed.Meanwhile,Transwell experiment found that the change of FAT10 had no significant effect on macrophage recruitment after HIF1α knockout.3.Flag-FAT10 and sh FAT10 plasmids were transfected into 266-6 and HEK293 T cells,or IFN-γ/TNF-α was given to stimulate endogenous FAT10 upregulation.WB experiment showed that after FAT10 interference,VHL protein expression increased,HIF1α decreased,and vice versa.However,Quantitative Real-time PCR results showed that FAT10 had no effect on VHL m RNA level.On the other hand,IP assay confirmed that HIF1α ubiquitination decreased when FAT10 was upregulated,whereas HIF1α ubiquitination increased when FAT10 was interfered.Then 266-6 cells were transfected with FAT10 overexpressed plasmids,VHL overexpressed plasmids and FAT10 and VHL overexpressed plasmids at the same time,and WB experiments showed that the expression of HIF1α protein in the group transfected with FAT10 and VHL overexpressed plasmids at the same time decreased compared with the group transfected with FAT10 overexpressed plasmids alone.On the other hand,the expression of HIF1α protein was increased in HEK293 T cells transfected with FAT10 interfering plasmid,VHL interfering plasmid,FAT10 interfering plasmid and VHL interfering plasmid respectively,and the results showed that the expression of HIF1α protein was increased in the group transfected with FAT10 and VHL interfering plasmid compared with the group transfected with FAT10 interfering plasmid alone.Finally,IP assay confirmed that HIF1α ubiquitination was increased in 266-6 cells transfected with both FAT10 and VHL overexpressing plasmids compared with the group transfected with FAT10 overexpressing plasmids alone,However,in HEK293 T cells,the level of HIF1α ubiquitin decreased in the FAT10 and VHL-interfered plasmid transfection group compared with the FAT10-interfered plasmid transfection group alone.4.WB results showed that FAT10 regulation of VHL expression was independent of UB.Then we add MG132 in 266-6 cells,WB results show that the change FAT10 for VHL protein expression had no effect.At the same time,the addition of actinomycin CHX to 266-6 cells blocked protein synthesis,and it was found that the half-life of VHL protein was significantly shortened in 266-6 cells transfected with FAT10 overexpressing plasmid.On the other hand,FAT10 was gradually overexpressed in wild-type 266-6 cells and FAT10 knockout 266-6 cells,and the effect of FAT10 on VHL was found in a dose-dependent manner.Through endogenous IP and exogenous IP experiments,it was found that FAT10 could bind to VHL in 266-6 cells,and the colocalization of FAT10 and VHL in 266 cells was observed by laser confocal microscopy.Then it was confirmed by IP experiment that FAT10 could make VHL FAT10 ylation,but FAT10 did not bind to VHL after mutating the site of FAT10 binding substrate.Finally,we further demonstrated that FAT10 regulation of VHL expression is critical in HIF1α ubiquitination in FAT10 knockout 266-6 cells by either TNF-α stimulation or FAT10 overexpression plasmid transfection.5.Through endogenous and exogenous IP experiments,it was found that FAT10 could bind to HIF1α in 266-6 cells.Meanwhile,the co-localization relationship between FAT10 and HIF1α in 266-6 cells was observed by laser confocal microscopy.Subsequently,it was confirmed by A498 cells with natural deletion of VHL gene that the ubiquitination level of HIF1α decreased with the gradual increase of FAT10 expression,and the binding of FAT10 to HIF1α increased gradually.We further altered the expression of RPN10 in 266-6 cells and interfered with UB to block the ubiquitin degradation pathway.We found that FAT10-HIF1α complexes and HIF1α protein expression did not change significantly after altered the expression of RPN10,while FAT10-p62 complexes and P62 protein still changed.Finally,we treated 266-6 cells with TNF-α/IFN-γ to stimulate the increase of endogenous FAT10 protein expression or exogenous FAT10 overexpressed plasmid was introduced into FAT10 knockout 266-6 cells.It was found that the protein expression of HIF1α increased with the increase of plasmid concentration,and the binding amount of FAT10 to HIF1α also increased with the increase of plasmid concentration.Conclusion: 1.FAT10 knockout can reduce macrophage infiltration in pancreatic tissue,thus alleviating acute pancreatitis.2.Inflammation-mediated FAT10 promotes HIF1α dependent recruitment of macrophages.3.FAT10 inhibited HIF1α ubiquitination by down-regulating VHL expression,and thus promoted HIF1α protein expression.4.FAT10 induces FAT10 ylation of VHL,which promotes the degradation of VHL protein.5.FAT10 can stabilize HIF1α and avoid degradation by proteasome.