PC1 C Terminal Tail Regulates E3 Ubiquitin-protein Ligase ?-TrCP And Its Potential Therapeutic Implication In ADPKD

Background : Autosomal dominant polycystic kidney disease(ADPKD)is a systemic disease with an incidence of 1/1000 to 1/400.It is the most common hereditary kidney disease,mainly caused by mutations in the PKD1 or PKD2 genes.It is characterized by bilateral multiple renal cysts formation and progressive enlargement of the total volume of the kidney,leading to urinary enrichment disorders,hypertension,polyuria,nocturia,pain,kidney stones,hematuria,infection and progressive loss of renal function.In Europe,one in every 10 patients with end-stage renal disease is caused by ADPKD,and in every 20 patients in the United States,Australia,and New Zealand has ADPKD.For a long time,the treatment and management of polycystic kidney disease has not progressed as rapid as other kidney diseases.Therefore,it is particularly important to find effective treatment for ADPKD.The ubiquitin-proteasome system(UPS)is an important protein degredation system responsible for the degradation of proteins involved in lots of activities,including cell cycle progression,apoptosis,DNA damage/repair,endocytosis,and drug resistance,angiogenesis and cell differentiation.Thus the UPS plays an important role in tumorigenesis and tumor survival.The ?-transduction repeat containing protein(?-TrCP)is a member of the SCF family and plays a significant role in the development of various tumors.There are few studies on UPS in autosomal dominant polycystic kidney disease.Therefore,this paper mainly discusses the role and regulation mechanism of E3 ubiquitin-ligase ?-TrCP in ADPKD.Througn Inhibiting UPS or specific inhibiting ?-TrCP,We observed the effect on cyst growth.This may provide a new idea for clinical treatment of ADPKD.Methods:First,we wanted to study the expression of ?-TrCP in polycystic kidney disease.We detected the renal tissue of patients with polycystic kidney disease and PKD mouse model and cyst-lining cells of human by western blot,immunohistochemistry and immunofluorescence.We found he expression of ?-TrCP in the PKD tissues and cyst-lining epithelial cells were elevated.Second,we were interested in the mechanisms of ?-TrCP up-regulation in polycystic kidney disease.We overexpressed STAT1 and inhibited STAT1 expression in cyst-lining epithelial cells.Then we detected the expession of ?-TrCP and the promoter activity of BTRC.Then we overexpressed PC1-CTT to detect ?-TrCP expression and promoter activity.We found that the expression of ?-TrCP can be up-regulated by PC1-CTT through activating the JAK2-STAT1 pathway.Next,we wanted to understand the role of ?-TrCP in the development of polycystic kidney disease.We found that the growth of the cysts were inhibited by the UPS inhibitor PS-341 in MDCK cells,and specifically knocking down of ?-TrCP in IMCD3 cells.?-TrCP act as an oncogenic protein in polycystic kidney disease.Next,we examined the proliferation and apoptosis after PS-341 treatment,as well as the morphology of cilia and the expression of PC2.We found that PS-341 inhibited proliferation,promoted apoptosis,and increased the length of cilia and the location of PC2 in cilia.However,the total amount of ?-TrCP substrates PDCD4 and PC2 did not change.This led us to explore the crosstalk between autophagy and the ubiquitin-proteasome pathway.We found that autophagy pathway was activated.After specifically knocking down of ?-TrCP,we found that the cilia became longer and the expression of PC2 in cilia increased.The expression of PDCD4 was up-regulated,and the autophagy pathway was not significantly changed showing that the specifically knocking down of ?-TrCP is more precisely than PS-341.Since there is no specific ?-TrCP inhibitor,we finally use PS-341 to treat the PKD mice to evaluate the effect of inhibiting UPS in polycystic kidney disease.Results:We found that the expression of ?-TrCP was up-regulated in patients and animal models of autosomal dominant polycystic kidney disease,and mainly localized in the nucleus.Next,we investigated the mechanism of high expression of ?-TrCP in human cyst-lining epithelial cells.We found that PC1-CTT can increase the expression of ?-TrCP by activating the downstream JAK2-STAT1 signaling pathway.The ubiquitin-proteasome system inhibitor PS-341 can inhibit the formation of vesicles in MDCK cells in a three-dimensional culture environment promoteing apoptosis and inhibiting cell proliferation.Specifically knocking down of ?-TrCP inhibits the formation of vesicles of IMCD3 cells.Knocking down of ?-TrCP in human cyst-lining epithelial cells increased the expression of PDCD4,cilia length and PC2 expression in cilia but not the total PC2 expession,indicating that down-regulation of ?-TrCP can promote cilia formation and functional PC2 entering into the cilia.Interestingly,the cilia become shorter and many may not even form normally when overexpress BTRC in human cyst-lining epithelial cells.When we treated the cystic cells with PS-341,we found that the autophagy pathway was alternatively activated.Neither PDCD4 nor PC2 changed but the cilia length and the expression of PC2 in cilia increased.We hypothesized that PS-341 prevent polycystic kidney disease by increasing the expression of functional PC2 in the cilia.Last,We administered the early-onset PKD mice PS-341 0.3 mg/kg intraperitoneally twice a week.The specimens were collected for renal function and cyst index and survival curve.We found that PS-341 significantly reduced cyst index,improved renal function and prolonged survival of the PKD mice.We further examined the proliferation,apoptosis and inflammation indicators in renal tissues,and we found that the proliferation of mouse kidney tissue was decreased,the apoptosis increased,and the inflammatory cell infiltration was significantly reduced after PS-341 treatment.Conclusions:(1)PC1-CTT promotes the expression of ?-TrCP by activating the JAK2-STAT1 pathway in autosomal dominant polycystic kidney disease;(2)?-TrCP is highly expressed and acts as a oncogenic protein in polycystic kidney disease;(3)specifically knocking down of ?-TrCP inhibits cyst growth by up-regulating PDCD4,increasing cilia length and the effective dose of PC2 in cilia;(4)PS-341 promotes apoptosis,inhibits cell proliferation,and ultimately inhibits cysts growth,possibly by increasing the cilia length and founctional PC2 in the cilia.(5)PS-341 significantly inhibits cystic growth,improve renal function,and prolong survival of PKD mice through inhibiting cell proliferation,promoting apoptosis,and reducing renal interstitial macrophage infiltration.(6)PS-341 alternatively activated the autophagy pathway,indicating that inhibition of UPS may not be a perfect strategy to prevent polycystic kidney disease.