The Role Of ICAT In Meangial Cells Pheontypic Transition By Regulating PPARγ Transcriptional Activity

BackgroudMore and more people are suffered from chronic kidney disease(CKD)and end-stage renal disease(ESRD)caused by diabetic nephropathy(DN)as our country developes.Owing to the complicated mechanism of DN,there is no effective therapeutic drug to now.Peroxisome proliferator-activated receptor γ(PPARγ)is the molecular target of thiazolidinediones drugs(TZDs).Researches indicated that TZDs benefited DN patients by lowering urine albumin excretion and protecting residual renal function.Previously we found the expression of β-catenin interacting protein-1(ICAT)is reduced in human mesangial cells(HMCs)cultured in high glucose.In recent years,interplays between PPARγ and the Wnt/β-caternin pathways have been found in malignant tumors.A report showed that β-catenin-ICAT protein complex could act as a coactivator in the nucleus to enhance AR-mediated transcription.So we propose a hypothesis that β-catenin-ICAT complex acts as a coactivator to activate peroxisome proliferator-activated receptor γ(PPARγ)and manipulate its transcriptional activity.Suppressed complex formation due to low ICAT expression leads to the lower PPARγ transcriptional activity in hyperglycemia.This finding may provide a new insight in elucidating the mechanism of DN.Methods1.HMCs were cultured in either 5.5(normal control,NC)or 30(high glucose,HG)mmol/L glucose medium.Gene expression level was evaluated by western blot and real-time PCR.2.PPARγ transcriptional activity was evaluated by luciferase assay in different conditions.3.Cell phenotype transition of HMCs was detected by the expression level of ?-SMA and fibronectin,as well as cck-8 assay.4.Protein-protein interation was predicted by bioinformatics prediction and tested by co-immunoprecipitation(co-IP).ResultsⅠ Gene expression level and PPARγ transcriptional activity in different concentrition of glucose1.There was no significant change of PPARγ expression in HMCs cultured in HG,while the proliferation rate of HMCs in HG was increased.2.ICAT expression level was reduced while β-catenin expression level was elevated in HMCs cultured in HG.3.Transcriptional activity of PPARγ was suppressed in HMCs cultured in HG compared with NC.ⅡThe relationship between overexpression/knockdown of ICAT/β-catenin and PPARγ transcriptional activity.1.Knockdown of ICAT in NC leads to decreased PPAR? transcriptional activity compared with either NC or a non-target siRNA group,PPAR? transcriptional activity was partly reversed in ICAT overexpression group compared with cells either in HG or transfected with green fluorescent protein(GFP),PPAR? transcriptional activity remained still low when kockdown of ?-catenin in HG.2.Overexpression of ICAT can partially reduce cell phenotypic transition markers(?-Sma and fibronectin)of HMCs in HG.3.Lower cell proliferation was tested in the ICAT overexpression group compared with HG.Ⅲ.The interaction between PPARγ,?-catenin and ICAT.1.PPARγ can bind to the NH2 terminal of the ?-catenin-ICAT complex without affecting its own interaction domain.2.Co-IP test showed PPARγ,?-catenin and ICAT can interact in HMCs.Conclusion1.HG induced lower ICAT but higher ?-catenin expression in HMCs.2.HG induced lower PPARγ transcriptional activity without affecting PPARγ expression.3.Lower expression of ICAT induced by HG leaded to lower PPARγ transcriptional activity and HMCs phenotypic transition.4.Overexpression ICAT in HG can increase PPARγ transcriptional activity and relieve HMCs phenotypic transition5.PPARγ,?-catenin and ICAT can interact as a protein complex in HMCs.