| Objective:The generation and maintenance of asymmetric polarity along the apico-basalaxis is a ubiquitous and fundamental requirement for many cellular processesincluding epithelial barrier function, cell migration, asymmetric cell division andmorphogenesis in tissues and organs such as the kidney. Establishment of epithelialapico-basal polarity is crucial for proper kidney development and function. It is ofgreat significance to understand the regulatory mechanism of renal epithelial cellpolarity and signal pathways. In this study, we sought to investigate the potentialmechanisms of PPARγ activation on apico-basal polarity and cell migration inrenal epithelial cells.Method:We use the well-established three-dimensional (3D) Madin-Darby canine kidney(MDCK) model to study the lumen formation and localization of polarity proteins atthe early stage of the process.The calcium switch model, immunofluorescence staining and measuring thetransmembrane electrical impedance are performed to identify the effects ofRosiglitazone in the establishment of epithelial apico-basal polarity including thedevelopment and maintenance of Apical Domains and the formation of tightjunction.Through the wound healing assays, we analyze the role of Rosiglitazone in cellmigration and localization of Par3and Occludin at the leading edge.We also detected polarity proteins expression of Par3and Occludin usingWestern Blotting method.Results:MDCK II cells were embedded in type I collagen gels and cultured for7days.Rosiglitazone (20uM) reduced the percentage of single central lumen cysts(66.20±7.69%in the DMSO group vs.18.74±6.26%in the Rosiglitazonegroups,t=44.46,P=0.005). There was a corresponding increase in the percentage ofmultiple lumen cysts (26.70±4.62%in the DMSO group vs.67.98±8.09%in theRosiglitazone groups, t=6.046,P=0.0263); the localization of Par3and Apkc were notabnormal; at the early stage of MDCKII cysts (2-5cells), Rosiglitazone inducedmislocalization of apical (Ezrin) and basolateral (E-cadherin) membrane proteins.We analyzed the repolarization process of MDCKII cell induced by a calciumswitch (CS),Rosiglitazone retarded apical membrane domain development in theearly phase of cell polarization (CS0h: the proportion of VACs in total cells,34.11±4.23%in control group vs42.36±3.87%in Rosiglitazone group; CS3h: theproportion of VACs in total cells,11.28±1.41%in control group vs28.44±2.30% in Rosiglitazone group); while during the maintenance phase of cell polarity, theapical domain retention was significantly affected Rosiglitazone group (CS3h: theproportion of cells retaining Ezrin at the cell periphery was61.24±4.59%in controlgroup vs12.38±2.69%in Rosiglitazone group, t=13.50,P=0.0054). Rosiglitazonegroup exhibited a profound delay in the formation of TJs which was monitored bystaining of the TJ proteins ZO-1;24hr after CS, however, there were no apparentdifferences between control and Rosiglitazone group; besides this, the developmentof the TER was significantly disturbed in Rosiglitazone group. We also verified Par3low expression on protein level.Wound healing assays were performed with control and Rosiglitazone group(the control MDCK cells showed37.63±2.50%wound closure17hr after wounding,but the Rosiglitazone group covered only23.07±4.86%of the control area,t=4.524,P=0.0106). Rosiglitazone impaired the leading edge localization of Par3andOccludin and change the morphology of cytoskeletal (F-actin). Meanwhile,Rosiglitazone down regulated Occludin expression on protein levels.Conclusion:In three-dimensional culture systems,Rosiglitazone disturbed apical proteindelivery to the plasma membrane, and eventually leaded to abnormal in theestablishment of apico-basal polarity which affected lumen formation in MDCKIIcell.Rosiglitazone was involved in the development and maintenance of apicaldomains and the formation of Tight Junctions.Rosiglitazone disturbed the leading-edge localization of polarity proteins Par3and Occludin and regulated cell migration. |
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