Study On The Role And Mechanism Of PAK4 In TGF-?-induced EMT In Renal Tubular Epithelial Cells

Objective: Chronic kidney disease(CKD)is a collection of the irreversible damage renal disease,eventually progress to end-stage renal disease(ESRD).Accounting for about 1.2% of children's kidney disease,CKD mortality rate was 9%.Once the disease progress to ESDR,it will produce a great impact on the growth,heart health,quality of life of children and has a high mortality rate.The epithelial-mesenchymal transition(EMT)of renal tubular epithelial cells has played a very important role in development in renal fibrosis of CKD.In numerous EMT regulation mechanism,TGF-beta signaling pathway is thought to be the main regulatory mechanism,and as the main factor promoting fibrosis,TGF-beta plays an important role in EMT of the renal tubular and glomerular podocyte.P21 activated protein kinase(PAK)is a kind of conservative serine/threonine protein kinase.Research shows that PAK4 is associated with EMT in tumor.It is an important target protein of Cdc42 and Rac.It can be activated by the growth factors and extracellular signals in GTPase dependent and independent singnaling pathways,involved in regulating the cytoskeleton,cell survival,mitosis and angiogenesis.So far,we have found that PAKs family has six members.It can be divided into two categories,according to the characteristic and similarity of structure,type I including PAK1,PAK2 and PAK3 and type II including PAK4,PAK5 and PAK6.PAK4 is the first member reported in type II family and it has been reported that it was highly expressed in the prostate,testicles and colon.In this study we use Transforming growth factor beta1(TGF-?1)to induce EMT of human renal tubular epithelial cells and to discuss the expressional changes of PAK4 in the EMT of renal tubular epithelial cells.Then we study the role and the mechanism of PAK4 in the process of TGF-?1 induced EMT of renal tubular epithelial cells.Methods: Regular cultivating human renal tubular epithelial cells(HK-2 cells),given different concentrations of TGF-?1(0,1ng/ml,2 ng/ml,5 ng/ml and 10 ng/ml)to stimulate HK-2 cells for 48 hours,or 5ng/ml TGF-?1 to stimulate HK-2 cells for different time(0,12,24,48 and 72 hours),morphological changes were observed under phase contrast microscope;the expressional changes in protein and mRNA of PAK4,E-cadherin,Fibronectin and Vimentin are tested using Western blot,RT-PCR and immunofluorescence assay;the changes of cellular migration ability of HK-2 were detected using Transwell Chambers method.According to the gene sequence specific primer design,specific primers were designed to amplify the PAK4 gene fragment,and Flag-PAK4 was transfected into HK-2 cells by the slow virus transfection method,and then the stable transfected cell line of overexpressed PAK4 was constructed.After 5 days of slow virus transfection,we tested the cell line using Western blot to confirm whether the stable cell line of overexpressed PAK4 was constructed successfully.In the steady cell line of overexpressed PAK4,the expressional changes in protein and mRNA of E-cadherin,Fibronectin and Vimentin are tested using Western blot,RT-PCR and immunofluorescence assay.Transwell Chambers was also used to detect the changes of cell migration ability.In addition,5 ng/ml TGF-?1 was given to the steady cell line of overexpressed PAK4 for 48 hours,then the application of phase contrast microscope observed the cell morphological changes of cells overexpressed PAK4.The Western Blot and RT-PCR were used to detect the expressional changes in protein and mRNA of E-cadherin,Fibronectin and Vimentin in each group induced by TGF-?1.Transwell Chambers were also used to detect the changes of the cell migration ability.In addition,the human PAK4 targeting sh RNA was designed and transfected into HK-2 cells by the slow virus transfection method and the stable transfected cell line was constructed with sh PAK4.After 5 days of slow virus transfection,we tested the cell line using Western Blot to confirm whether the stable cell line of silenced PAK4 was constructed successfully.In the steady cell line of silenced PAK4,the expressional changes in protein and mRNA of E-cadherin,Fibronectin and Vimentin are tested using Western Blot,RT-PCR and immunofluorescence assay.Transwell Chambers was also used to detect the changes of cell migration ability.In addition,5 ng/ml TGF-?1 was given to the steady cell line of silenced PAK4 for 48 hours,then the Western Blot,RT-PCR and immunofluorescence assay were used to detect the expressional changes in protein and mRNA of E-cadherin,Fibronectin and Vimentin in each group induced by TGF-?1.Transwell Chambers were also used to detect the changes of the cell migration ability.Western Blot was used to detect the changes of the beta-catenin in the stable cell line of silenced PAK4,and the changes of the location of beta-catenin in the stable cell lines of silenced PAK4 were detected by the immunofluorescence assay.Results:1.The HK-2 cell deform to shuttle,thin,long,the cell space increases,and number of cells decrease under the same view after stimulate by TGF-?1.2.TGF-?1 downregulate the expression of E-cadherin protein and mRNA and upregulate the expression of Fibronectin and Vimentin protein and mRNA using time dependent and dose dependent method.The optimal duration is 48 hours and the best concentration is 5 ng/ml.3.The expression of PAK4 increases in the process of EMT of HK-2 cell stimulated by TGF-?1 and with the increase of dose and time of stimulating of TGF-?1 the expression of PKA4 increases.But the expression of mRNA dose not change.4.With the increase concentration of TGF-?1,HK-2 cell migration ability enhances.5.Use slow virus transfection method to construct a PAK4 expression and PAK4 silence stable cell line successfully in HK-2 cells.6.The HK-2 cell deform to shuttle,thin,long,the cell space increases,and number of cells decrease under the same view after overexpression of PAK4.7.The expression of E-cadherin protein and mRNA decrease and the expression of Fibronectin and Vimentin protein and mRNA increase after overexpression of PAK4.8.The migration ability of HK-2 cell increases and the migration ability of TGF-?1 induced HK-2 cells further increase after overexpression of PAK4.9.The expression level of E-cadherin protein and mRNA further decreases and The expression level of Fibronectin and Vimentin protein and mRNA further increases after overexpression of PAK4.10.The expression of E-cadherin protein and mRNA increases and The expression of Fibronectin and Vimentin protein and mRNA decreases after silence of PAK4.11.The migration ability of HK-2 cell decreases and the migration ability of TGF-?1 induced HK-2 cells further decreases after silence of PAK4.12.The expression of TGF-?1 induced E-cadherin protein and has on change,but the expression of mRNA increases,the expression of Fibronectin and Vimentin protein and mRNA decreases after silence of PAK4.13.The expression of TGF-?1 induced ?-catenin further increased after silence of PAK4.14.?-catenin protein transports from cell membrane to the nucleus after the immunofluorescence visible overexpression of PAK4.Conclusion: 1.TGF-?1 can induce the EMT of HK-2 cells and is in dose dependent and time dependent relationgship with E-cadherin?Fibronectin and Vimentin.With the increase of TGF-?1,HK-2 cells migration ability enhances.2.TGF-?1 can induce the EMT of HK-2 cells.PAK4 also take part in the process.The expression of PAK4 is time and dose dependent with TGF-?1.But the expression of mRNA does not change.3.The PAK4 induce EMT of HK-2 cells independently.4.The overexpression of PAK4 may promote EMT of TGF-?1 induced HK-2 cells.5.Silence PAK4 can inhibit EMT of TGF-?1 induced HK-2 cells.6.PAK4 promote EMT of TGF-?1induced HK-2 cells by the ?-catenin pathway.