Role Of FHL2 In Regulating Interstitial Fibrotic Through ?-catenin Signaling Pathway

Objectives:To investigate the role of FHL2 in regulating interstitial fibrotic through?-catenin signaling pathway.Methods:1.The FHL2 mRNA and protein expression in renal interstitial fibroblasts(NRK-49F)which were stimulated with transforming growth factor ? 1(TGF-? 1)were analyzed by RT-PCR and Western Blot approach.2.FHL2 plasmid or small interfering RNA(SiRNA)were transfected to upregulate or downregulate the FHL2 protein level in NRK-49F cells in order to observe the fibrosis related proteins expression through Western Blot and immunofluorescene.3.FHL2 plasmid was transfected to upregulate the FHL2 protein level in NRK-49F for 48 hours,then use nuclear and cytoplasmic extraction regents to stepwise separate cytoplasmic and nuclear extracts to observe the activation of ?-catenin.The interaction of FHL2 and ?-catenin were revealed by co-immunoprecipitation,Western Blot.To observer the transcriptional regulation of ?-catenin by FHL2 in NRK-49F,TOP flash approach were applied.4.The animal model of renal interstitial fibrosis was induced by unilateral ureteral obstruction(UUO)in CD-1 mice.Age and weight-matched sham-operated mice were used as normal controls.The expression of FHL2 and active-?-catenin in normal and fibrotic kidneys after obstructive injury were analyzed by Western Blot and immunohistochemical.5.The animal model of renal interstitial fibrosis was also induced by unilateral ureteral obstruction(UUO)in mice with interstitial fibroblast-specific ablation of FHL2(Fibro-FHL2-/-mice),whereas age and gender matched FHL2-floxed littermates were considered as control(Fibro-FHL2+/+ mice).The severity of fibrosis after the injury were analyzed by Western Blot and immunohistochemistry technology.Results:1.TGF-?1 induced FHL2 mRNA and protein expression in rat renal interstitial fibroblasts(NRK-49F)in a time-or dose-dependent fashion,.2.Overexpression of FHL2 increased a-smooth muscle actin(?-SMA)?CollagenI and Fibronectin(FN)expression in NRK-49F cells,whereas knockdown of FHL2 partially reduced TGF-?1-mediated a-SMA?CollagenI and FN expression.Immunofluorescene showed a similar result.3.P-catenin nuclear translocation was promted wheareas cytoplasmic translcation had no obvious change when NRK-49F cells upregulated FHL2 protein.Western Blot and TOP flash show that FHL2 can regulate the activity of ?-catenin.4.In a mouse model of obstructive nephropathy,FHL2 expression increased in a time-dependent manner.Immunofluorescence showed that FHL2 increased in fibrotic renal tissue.5.Western blot and immunohistochemical showed that the expression of a-smooth muscle actin(a-SMA)?CollagenI and FN were obviously reduced in Fbro-FHL2-/-mice compared to FHL2+/+mice.The comparison of interstitial fibrotic area and total collagen content showed similar results.