The Role Of SOCE And The Mechanism Involved In The Development Of Acute Pancreatitis

Background Acute pancreatitis?AP?is an acute inflammatory process of the pancreas with its self-digestion,edema,hemorrhage and necrosis as the main characteristics.The pathogenesis of AP has not been established completely.Ca²⁺overload is a key contributor to pancreatic acinar cell injury due to prolonged and global intracellular Ca²⁺concentration([Ca²⁺]_i)elevation that leads to pancreatic inflammation,edema and autophagic vacuolization,all of which are typical pathological features of AP.Store-operated calcium?SOC?channel is widely distributed in non-excitable cell and sustaining Ca²⁺entry will probably depend on the SOC channels in the initiation of AP.Recently,calcium release-activated calcium channel?CRAC?composed of stromal interacting molecule 1?STIM1?and Orai1 was identified as a typical SOC channel.However,the relationship between AP and CRAC-mediated store-operated calcium entry?SOCE?and the downstream mechanisms of SOCE in the development of AP are still unclear.Vacuolization is one of typical pathological features of AP and previous studies showed that the majority of vacuoles were autophagic in origin.Autophagy is considered an essential cause that induce inappropriate activation of trypsinogen to trypsin within pancreatic acinar cell.However,the relationship of Ca²⁺overload induced by SOCE in pancreatic acinar cells and autophagy need further investigation.Calcineurin?CaN?is a Ca²⁺/calmodulin?Ca M?-dependent protein phosphatase that can be activated by the[Ca²⁺]_i elevation in some physiological or pathological conditions.CaN activation dephosphorylates its target proteins and regulates physiological or pathological procedure.Transcription factor EB?TFEB?is a target protein of CaN,which activation regulates the activation of autophagy.However,it is not reported whether SOCE regulates the formation of autophagic vacuolization via CaN-activated TFEB activation.Object The object of this study is to morphologically and functionally investigate the relationship between STIM1-Orai1-mediated SOCE and the formation of vacuoles in AP by constructing the model of calcium overload in pancreatic acinar cells and animal pancreatitic model using caerulein,and further to explore the molecular mechanism of SOCE-induced pancreatic vacuolization in AP.Methords and Results Orai1 mediated the caerulein-induced SOCE in pancreatic acinar cells and promoted the formation of autophagic vacuoles.Pancreatic acinar cells were acutely dissociated and Western blotting showed that pancreatic acinar cell endogenously express STIM1 and Orai1.Confocal calcium imaging showed that caerulein induced SOCE which was blocked by Orai1 inhibitor GSK7975A.HE staining showed that GSK7975 A significantly reduced the severity of inflammation,edema and vacuolation in caerulein induced AP.Caerulein triggers SOCE via promoting the interaction between STIM1 and Orai1 To gain visualized evidence of whether caerulein can trigger SOCE via promoting the interaction between STIM1 and Orai1,the plasmids of CCK-A receptor?CCKAR?were constructed in the present study,which function was validated by confocal calcium imaging.The plasmids CCKAR-CFP,STIM1-YFP and Orai1-mCherry were co-overexpressed in 293 T cells,and live cell imaging showed that caerulein stimulation resulted in the appearance of distinct STIM1 puncta and induced the co-localization between STIM1 and Orai1.CaN mediated the caerulein-induced pancreatic injury To investigate the relationship between CaN activation and AP,FK506 was used to inhibit the CaN activation in vivo.HE staining showed that CaN inhibition significantly reduced the severity of inflammation,edema and vacuolation in caerulein induced AP.CaN activation depends on caerulein-induced SOCE Nuclear factor of activated T-cells?NFAT?was usually used to detect the activation of CaN.To clarificate the relationship of SOCE with CaN activation,the plasmids NFAT1-GFP and CCKAR-m Cherry in Hela cells were co-expressed in Hela cells.Confocal live cell imaging was used to dynamically observe the subcellular distribution of NFAT,and results showed that caerulein induced the NFAT nuclear translocation,which was arrested by removing the extracellular Ca²⁺or Orai1 inhibition or CaN inhibition.Calcineurin activity of pancreatic acinar cells was also analyzed the use of caerulein with/without FK506 or EGTA or GSK7975 A,and data showed that both removal of extracellular Ca²⁺and blocking Orai1 can inhibit the activation of CaN,which were consistent with the result obtained with CaN inhibitor.These data indicate that CaN activation depends on caerulein induced SOCE.The increase of autophagic vacuoles in AP may be caused by TFEB activation To obseve the level of autophagy before and after AP,LC3?and SQSTM1/P62 were detected by Western blotting,and data showed that the levels of LC3?and SQSTM1/P62 were significantly increased in AP.To further measure the mRNA level of LC3?and SQSTM1/P62,Realtime-PCR was used and data showed that both mRNAs were increased in AP.TFEB overexpression activated the transcription of autophagy-related genes?such as LC3?and SQSTM1/P62?and induced the autophagy activation.To explore the relationship of TFEB with autophagy activation in AP,we measured the level of TFEB by western blotting and found that the level of TFEB was significantly increased in AP.To further verify whether TFEB was led to nuclear tranlocation in caerulein-AP,we measured the level of cytosolic and nulear TFEB in pancreatic acinar cells.Data showed that the levels of nuclear TFEB was increased,while the cytosolic TFEB was also increased.SOCE-induced CaN activation regulates TFEB nuclear translocation To further confirm the relationship between caerulein and TFEB nuclear translocation,the CCKAR-mCherry and TFEB-EGFP plasmids were co-overexpressed in Hela cells and AAV-TFEB-EGFP was used to infect pancreatic acinar cells.Confocal live cell imaging showed that caerulein promoted the translocation of TFEB-EGFP from cytoplasm to nucleus.To expolre the relationship between TFEB nuclear translocation and CaN activation,theCCKAR-mCherryandTFEB-EGFPplasmidswere co-overexpressed in Hela cells by electroporation and AAV-TFEB-EGFP was used to infect pancreatic acinar cells.Confocal live cell imaging showed that inhibition of CaN or SOCE by FK506 or GSK7975 A obviously reduced the TFEB translocation to nucleus.Furthermore,pancreatic acinar cells were acutely dissociated and pretreated with FK506 or GSK7975 A before stimulating with supramaximal caerulein,Western blotting showed that both FK506 and GSK7975 A significantly inhibit the nuclear translocation of TFEB.All these data indicate that SOCE activated CaN can induce dephosphorylation and nuclear translocation of TFEB in AP.CaN inhibition reduced the formation of autophagosome To observe the effects of CaN activation in the formation of autophagosome in AP,pancreatic acinar cells were acutely dissociated and pretreated with FK506 before stimulating with supramaximal caerulein,Western blotting was used to detect the protein level of LC3?,and results showed that FK506 attenuated the increase of LC3?that induced by caerulein.Because CaN activation was regulated by STIM1-Orai1-induced SOCE,GSK7975 A was used to inhibit Orai1 in pancreatic acinar cells,and Western blotting results showed that GSK7975 A also attenuated the caerulein-induced increase of LC3?.Conclusion Caerulein induces SOCE by promoting the interaction between STIM1 and Orai1,and leading to calcium overload in pancreatic acinar cells;SOCE-induced calcium overload activates CaN,which in turn leads to the activation of TFEB and results in autophagic vacuolization in AP.