Par3-aPKC Interact With Vangl2 And Involve In Regulating E-cadherin-based Adherent Junctions And Epithelial Tubulogenesis

ObjectiveCell polarity is defined as morphologic and structurally asymmetric organization caused by the asymmetric distribution of such as intracellular components.organelles and cytoplasmic determinants.Epithelial cells,which typically form tubular structures during development,are specialized polar cells that form the tissues such as neural tube and digestive tract.Epithelial tissues exhibit two types of polarity:the apical-basal polarity(ABP)and planar cell polarity(PCP).Both ABP and PCP polarity pathways are illuminated to be complex,consisting of several modules of interacting protein by genetic and molecular analyses.Among ABP polarity pathway.Par3 is the most important component.Defects in Par3 are associated with several aberrant tubulogenesis such as polycystic kidney.Our previous studies showed that rare deleterious PARD3 variants were implicated in the pathogenesis of human cranial neural tube defects.Vangl2 is the most important key protein in PCP pathway.Similarly,deficiency of Vangl2 causes abnormal closure in neural tube.E-cadherin is the major adherent junctions molecule and is the basis of the establishment and maintenance of epithelial cell polarity,which abnormal distribution also caused defective closure in neural tube.In most previous description,the apical-basal polarity(ABP)and planar cell polarity(PCP)are considered separately.Several studies in Drosophila and Xenopus oocytes have confirmed the crosslink between components of apical-basal polarity(ABP)and Vangl2 of planar cell polarity(PCP).Yet.the interaction between two pathways,particularly the interaction between Par3 and Vangl2,has not been reported in mammalian.This study investigated whether there was a direct molecular crosslink between Par3-aPKC and Vangl2 in mammalian epithelial cells,then,explored the regulation of Par3 and Vangl2 to E-cadherin-based cellular adherent junctions.In addition,we assessed the involvement of the expression and distribution of Vangl2,Par3 and E-cadherin in the morphogenesis ofcysts.This study intends to explore the pathogenesis mechanism of abnormal tubulardiseases through the futher understanding of the role of polarity molecules in the process of tubulogenesis.Methods1.First,to characterize Par3-aPKC and Vangl2 localization in tubular epithelial cell,we set out to examine the localization of Vangl2 and Par3-aPKC at E10 embryonic mouse neuroepithelial cells and MDCK cells in three-dimensional(3D)cultures by Immunofluorescence(IF);we examined co-localization of Vangl2 and Par3-aPKC in 2D MDCK cells;To investigate whether Vangl2 can physically interact with Par3 or aPKC,we performed co-immunoprecipitation(COIP)and GST-pulldown experiments.2.We established MDCK cell clones with diminished Par3 and Vangl2 expression by shRNA-induced lentiviral infection,and investigated the expression and localization of Par3/Vangl2/aPKC by real-time PCR,Immunofluorescence(IF)and western blotting,confirming the regulation relationship between Par3 and Vangl2.3.Based on the importance of E-cadherin-based adherent junctions during tubulogenesis,we explored the regulation of Par3 and Vangl2 to E-cadherin-based adherent junctions in Par3-knockdown MDCK cells using IF,COIP,Transwell and wound healing assay.4.Using in vitro 3D cultures of MDCK cells which provide an ideal model system to study tubular morphogenesis in Par3-knockdown MDCK cells,we assessed the localization and outcomet of Par3,Vangl2 and E-cadherin in cyst formation.Results1.Vangl2 and Par3-aPKC localized and accumulated at the apical domain of the tubular epithelial cell.Par3-aPKC interacted and also colocalized with Vangl2 in mammalian epithelial cells.2.Par3 knockdown didn't affect the expression of Vangl2,whereas lead to its aberrant cellular localization.3.Par3,Vangl2 and E-cadherin can form protein complex in the mammalian epithelial cells.Both Par3 and Vangl2 involved in the regulation of E-cadherin-based adherent junctions.4.Knockdown of Par3 of MDCK cells induced aberrant cysts including multiple lumens and apical expansion lumens under 3D culture model,as well as changed distribution of Vangl2 and E-cadher in the lumen structure.5.The normal expression and distribution of Par3 and Vangl2 involved in epithelial tube formation.Conclusion1.The planar cell polarity key protein Vangl2 interacted with the apical-basal polarity protein complex Par3-aPKC.Par3 regulated the subcellular localization of Vangl2 in mammalian epithelial cells.2.Depletion of Par3 weakened E-cadherin-based adherent junctions,induced a decreased distribution of Vangl2 and E-cadherin along the apical membrane.Depletion of Par3 also affected the Vangl2/E-cadherin complex formation and their distributionsin the 3D lumen structure.The disturbed interaction between Par3,Vangl2 and E-cadherin along the apical domain and the following dis-localization in mammalian epithelial cells are the potential pathogenesis during abnormal tube formation.