The Role Of Polycystin 2-Scribble-YAP Pathway In Cyst Formation Of Autosomal-Dominant Polycystic Kidney Disease

Background and objective:Autosomal-dominant polycystic kidney disease?ADPKD?is the most common inherited fatal cystic kidney disease.It is caused by germline mutations in PKD1 or PKD2,which encode for proteins polycystin-1?PC1?and polycystin-2?PC2?,respectively.Previous studies have demonstrated PC1 and PC2expression in the apical monocilia,cytoplasmic endoplasmic reticulum/Golgi membranes and the basolateral cell surface,where they may be essential in regulating the function of cell membranes.The apical–basolateral polarity defects are common features in ADPKD and have been recognized to be able to facilitate the initiation and progression of ADPKD.Apical–basolateral polarity is organized by evolutionarily conservative complexes assembled around the proteins PAR,Crumbs,and Scribble,respectively.The Scribble complex,consisting of Scribble,Discs large?DLG?,and Lethal giant larvae?LGL?,plays an important role in the determination of basolateral membranes in epithelial cells.Scribble contains 16 leucine-rich repeats?LRRs?domains in its amino-terminus and 4PSD95/DLG/ZO-1?PDZ?domains in its C-terminus,which are protein–protein interaction modules that may have important roles in facilitating cell signaling by acting as a scaffold.A previous study has found that the knockdown of Scribble in zebrafish with morpholino?MO;scrib morphants?could induce pronephric cyst formation,indicating that Scribble may play an important role in regulating cyst formation.However,little is known about the role of Scribble in ADPKD.Recently,some studies suggest that Scribble could induce phosphorylation of Yes-associated protein?YAP?by large tumor suppressor kinase?LATS kinase?through the activation of Hippo signaling,which would subsequently prevent the nuclear localization of YAP.Hippo signaling pathway is inactivated in Pkd1 knockout mouse models and ADPKD patients with translocation of cytoplasmic YAP into the nucleus.YAP nuclear accumulation has been demonstrated as a promoter for cell proliferation in tumorigenesis.Alternatively,the previous study also reported that cytoplasmic YAP could inhibit tumor growth by regulating the Wnt/?-catenin signaling pathway.However,the role of cytoplasmic and nuclear YAP in regulating cyst formation in ADPKD remains obscure.Herein,we performed a series of experiments to elucidate the role of Scribble and cytoplasmic YAP in cyst formation,and Scribble could influence cyst formation in ADPKD by regulating nucleocytoplasmic shuttling of YAP.Methods:?1?The expression and localization of Scribble in ADPKD zebrafish model?pkd2 morphants?and cell model(mouse Pkd2^-/-cells)were detected by Western blot,immunofluorescence and RT-PCR analyses.Microinjection techniques were used to investigate the role of Scribble and its different domains?Scribble-LRR and Scribble-PDZ?in regulation of prerenal cyst formation in pkd2 morphants.?2?We used the CRISPR/Cas9system to generate the zebrafish scrib mutants,and observe whether the mutant fish had a polycystic kidney-related phenotype.?3?The expression of Hippo pathway molecules?pLATS1,LATS1,pYAP and YAP?were detected by Western blot,immunofluorescence,and RT-PCR in zebrafish scrib mutants and human Scribble knockdown renal tubular epithelial cells.?4?The expression and localization of pLATS1,LATS1,pYAP,and YAP were detected by Western blot,immunofluorescence,and RT-PCR analyses in pkd2morphants and Pkd2^-/-cells;Transgenic fish Tg?Tolcdh17:yap-gfp?,Tg?Tolcdh17:yapS87A-gfp?and Tg?Tolcdh17:yapS87D-gfp?were generated to observe the localization of different types of YAP?YAP,YAPS87A and YAPS87D?in zebrafish prerenal epithelial cells;Microinjection was performed to observe the effect of different types of YAP on the prerenal cysts formation in pkd2 morphants.Verteprofin treatment was performed to observe the effect of antagonizing nuclear YAP on prerenal cysts formation in pkd2morphants.?5?We generated lats1 mutants and yap mutants in zebrafish and observed whether the mutant fish had a prerenal cyst phenotype.?6?Scribble adenovirus was overexpressed in Pkd2^-/-cells to observe whether Scribble could regulate the expression of pLATS1,LATS1,pYAP and YAP in ADPKD;we overexpressed Scribble,Scribble-LRR and Scribble-PDZ in human renal tubular epithelial cells and observed which domain determined the subcellular localization of YAP;scrib morphants were generated by morpholino techniques,and different types of YAP?YAP,YAPS87A and YAPS87D?were overexpressed in scrib morphants to investigate their effect on the prerenal cyst formation.Results:?1?Scribble was down-regulated in pkd2 morphants and Pkd2^-/-cells.?2?Scribble was localized on the basolateral membrane of zebrafish prerenal epithelial cells;Scribble was co-localized with PC2 on the cell membrane.?3?Overexpression of Scribble and Scribble-PDZ could inhibit the prerenal cyst formation in pkd2 morphants,while overexpression of Scribble-LRR had no similar effect.?4?The zebrafish scrib mutant exhibited pronephric duct dilation;knockout of scrib could aggravate the prerenal cyst phenotype in pkd2 morphants.?5?The levels of pLATS1 and pYAP were down-regulated in zebrafish scrib mutants and human Scribble knockdown renal tubular epithelial cells;cytoplasmic YAP was translocated into the nucleus in prerenal epithelial cells of scrib mutant.?6?The levels of pLATS1 and pYAP were down-regulated in pkd2 morphants and Pkd2^-/-cells;cytoplasmic YAP was decreased,while nuclear YAP was increased in pkd2morphants and Pkd2^-/-cells.?7?The YAP and YAPS87D were localized in the cytoplasm of the prerenal epithelial cells of the transgenic fish,while the YAPS87A was localized in the nucleus.?8?Overexpression of YAP and YAPS87D could inhibit the prerenal cyst formation in pkd2 morphants,while overexpression of YAPS87A had no similar effect;Verteprofin treatment had no effect on reducing the polycystic kidney-related phenotype in pkd2 morphants.?9?The zebrafish lats1 mutant did not have a polycystic kidney-associated phenotype,but knockout of lats1 aggravated the prerenal cyst formation in pkd2 morphants.?10?The zebrafish yap mutants exhibited cystic kidneys;overexpression of YAP and YAPS87D could inhibit the prerenal cyst formation in yap mutants,while overexpression of YAPS87A had no similar effect.?11?Overexpression of Scribble in Pkd2^-/-cells could effectively increase the levels of pLATS1 and pYAP.?12?Overexpression of Scribble and Scribble-PDZ promoted the nuclear YAP to translocate into the cytoplasm,whereas overexpression of Scribble-LRR had no similar effect.?13?scrib morphants exhibited cystic kidneys;overexpression of YAP and YAPS87D could inhibit the prerenal cyst formation in scrib morphants,while overexpression of YAPS87A could promote the prerenal cyst formation in scrib morphants.Conclusions:?1?Scribble and cytoplasmic YAP play an important role in the regulation of cyst formation in ADPKD;?2?Scribble and cytoplasmic YAP are critical for the development of prerenal tubules and prerenal cyst formation in zebrafish;?3?Scribble influences cyst formation in ADPKD by regulating nucleocytoplasmic shuttling of YAP.