The Role Of Hsp90/Cdc37 Complex In Regulating Pancreatic Stellate Cells In Pancreatic Fibrosis

Background:Chronic pancreatitis(CP)increases risk of pancreatic cancer,pancreatic fibrosis is a main pathological feature of CP.Transforming growth factor-?1(TGF?1)induces the activation and extracellular matrix(ECM)synthesis of pancreatic stellate cells(PSCs),and transforming growth factor-? receptor(TGF?R)-mediated Smad and non-Smad signaling pathways play an important role in fibrotic disease,heat shock protein 90(Hsp90)is involved in the regulation of TGF?R stability,cell division cycle protein 37(Cdc37)is an important cochaperone of Hsp90,the Hsp90/Cdc37 complex formed by Cdc37 and Hsp90 prevents the degradation of unstable kinase.At present,the association between Hsp90 and pancreatic fibrosis has not been reported in CP,the relationship between Hsp90/Cdc37 complex and TGF?R-mediated Smad and non-Smad signaling pathways is not clear in PSCs,therefore,this study observed the relationship between Hsp90 and pancreatic fibrosis,and explored the mechanism of Hsp90/Cdc37 complex in regulating biological functions of PSCs.1 The association between serum heat shock protein 90? and chronic pancreatitis Objective: To evaluate the expression of Hsp90 in pancreatic tissues of CP mice,and observe the relationship between serum Hsp90 expression and CP.Methods: The model of CP mice was established by caerulein with intraperitoneal injection.Pancreatic tissues were collected,and were embedded with paraffin,paraffin sections were used for immunohistochemical staining to observe the expression of Hsp90 in pancreatic tissues.Proteins were extracted from pancreatic tissues,and the Hsp90 expression of pancreatic tissues was determined by western blots.Peripheral blood was collected,and western blots were used to determine the expression of serum Hsp90.Results: Immunohistochemical staining showed that Hsp90 was widely expressed in pancreatic tissues.Immunohistochemical staining and western blots showed that the expression of Hsp90 was increased in pancreatic tissues of CP mice.Western blots showed that the expression of serum Hsp90? was increased in CP mice.Conclusions: Serum Hsp90?levels are increased in CP mice,serum Hsp90? may be associated with CP.However,based on some shortcomings of the study in part,the current conclusions need to be further confirmed.2 Heat shock protein 90 inhibitor 17 AAG ameliorates pancreatic fibrosis in mice with chronic pancreatitis Objective: To observe the effects of Hsp90 inhibitor 17 AAG on pancreatic fibrosis in caerulein-incuded CP mice.Methods: Mice were divided into control group,high-dose 17 AAG control group,caerulein group,low-dose 17 AAG treatment group and high-dose 17 AAG treatment group.Pancreatic tissues were collected,and were embedded with paraffin,paraffin sections were used with immunofluorescent staining for inflammatory cells and ?-SMA in pancreatic tissues.The RNA of pancreatic tissues were extracted,q RT-PCR was used for gene quantification of alpha-smooth muscle actin(?-SMA),Collagen I(Col I),Fibronectin(Fn)and inflammatory cytokines.Proteins of pancreatic tissues were extracted,western blots were used for ?-SMA,Col I and Fn.Results: The Hsp90 inhibitor 17 AAG effectively reduced weight loss of CP mice.The results of q RT-PCR showed that the expression of tumor necrosis factor-?(TNF-?),interleukin-1?(IL-1?)and interleukin-6(IL-6)was significantly increased in pancreatic tissues of CP mice,Hsp90 inhibitor 17 AAG significantly reduced the expression of these inflammatory cytokines.Immunofluorescent staining showed that the infiltration of neutrophils,T lymphocytes and macrophages was observed in pancreatic tissues of CP mice,Hsp90 inhibitor 17 AAG reduced the infiltration of neutrophils,T lymphocytes and macrophages in pancreatic tissues of CP mice.Double immunofluorescence of ?-SMA and SM22 a indicated that Hsp90 inhibitor 17 AAG reduced the expression of ?-SMA in PSCs of CP mice.q RT-PCR and western blots revealed that Hsp90 inhibitor 17 AAG effectively reduced the expression of ?-SMA,Col I and Fn in pancreatic tissues of CP.Conclusions: Hsp90 inhibitor 17AAG ameliorates CP-induced pancreatic fibrosis by reducing pancreatic inflammation of CP mice,Hsp90 may be a new target for future anti-pancreatic fibrosis therapy in CP.3 The mechanism of Hsp90/Cdc37 complex in regulating pancreatic stellate cells activity and extracellular matrix accumulationObjective: To explore the mechanism of Hsp90/Cdc37 complex in regulating the activity and ECM synthesis of PSCs.Methods: Primary PSCs were isolated,and TGF?1-induced PSCs were used as a vitro model.Proteins were extracted from cells,western blots were used to determine the expression of ?-SMA,Col ? and Fn,et al,co-immunoprecipitation was used to assess the interaction between Hsp90 and TGF?R?.The RNA were extracted from cells,q RTPCR was used to evaluate the expression of TGF?RII m RNA.Results: Western blots indicated that TGF?1 significantly increased the expression of ?-SMA,Col I and Fn in PSCs,Hsp90 inhibitor 17AAG effectively inhibited TGF?1-induced expression of ?-SMA,Col I and Fn in PSCs,Hsp90 inhibitor 17 AAG had no effects on the expression of Hsp90 in PSCs.The PI3 K inhibitor wortmannin significantly inhibited TGF?1-induced phosphorylation of Akt and GSK-3? in PSCs.Hsp90 inhibitor 17 AAG significantly inhibited TGF?1-induced phosphorylation of Smad2/3,Akt,and GSK-3? in PSCs.The results of q RT-PCR indicated that Hsp90 inhibitor17 AAG had no significantly effects on the expression of TGF?RII m RNA in PSCs,western blots revealed that 17 AAG significantly reduced TGF?1-induced enhancement of TGF?RII expression in PSCs.Celastrol is a specific inhibitor that can effectively reduce the interaction between Hsp90 and Cdc37,the results of q RT-PCR showed that Celastrol had no significantly effects on the expression of TGF?RII m RNA in PSCs,western blots revealed that Celastrol significantly inhibited TGF?1-induced enhancement of TGF?RII expression in PSCs.After treatment with proteasome inhibitor MG132 for PSCs,western blots demonstrated that MG132 prevented Hsp90 inhibitor 17AAG-induced inhibition of Smad2/3 and PI3K/Akt/GSK-3? phosphorylation,and MG132 prevented the degradation of TGF?RII induced by17 AAG.The co-immunoprecipitation revealed that Hsp90 inhibitor 17 AAG reduced the interaction between Hsp90 and TGF?RII,increased the expression of ubiquitinated TGF?RII in PSCs,Celastrol resulted in reduced binding of Hsp90 with TGF?RII in PSCs.Conclusions:The Hsp90/Cdc37 complex effects the activation and ECM synthesis of PSCs via regulating TGF?R?-mediated Smad and non-Smad signaling pathways by ubiquitination-proteasome pathway.