The Effect Of TGF-β1-mediated Rho/ROCK Signaling Pathway On Tight Junctions Of HK-2 Cells

Objective:To investigate the effect of TGF-β1-mediated Rho/ROCK signaling pathway on tight junctions of HK-2 cells.Methods:(1) Renal tubular epithelial cells (HK-2) were stimulated by 5ng/ml TGF-β1 for different time. Western Blot were used to observe protein expression of RhoA (included active RhoA and total RhoA);Real time PCR, Western Blot and immunofluorescence assay were used to detect the mRNA and protein expression of Occludin and ZO-1.(2) HK-2 cells were randomly divided into 3 groups:group A(control), group B (TGF-β1,5ng/ml), group C (TGF-β1,5ng/ml+Y-27632,an ROCK inhibitor, 10μM), the mRNA and protein expression levels of Occludin and ZO-1 were measured by Real time PCR, Western Blot and immunofluorescence assay, respectively.Results:(1) Western Blot showed that, the protein expression of total RhoA in HK-2 cells did not change significantly after TGF-β1 stimulation,;active RhoA was remarkly increased 15min (2.82+0.57 folds), peaking at 1h(4.06±0.54 times),and dropping after 4h(3.44±0.31 times), and at 12h was still higher than 0 time point, which was (3.01±0.44)times, all of p values compared with 0 time point were less than 0.05.The results of Real time PCR showed that, the mRNA levels of Occludin and ZO-1 in the TGF-β1 stimulated groups compared with in the control group were decreased in a time-dependent manner (24h,48h P<0.05).Western Blot and immunofluorescence respectively showed that, protein levels of Occludin and ZO-1 in the TGF-β1 stimulated groups compared with in the control group were decreased in a time-dependent manner (24h,48h P<0.05).(3)The results of Real time PCR showed that, the mRNA of Occludin and ZO-1 in TGF-β1+Y-27632 groups compared with TGF-β1 stimulated groups had obvious increase (P<0.05),and Western Blot and immunofluorescence also showed that protein expression levels of Occludin and ZO-1 in TGF-β1+Y-27632 groups compared with TGF-β1 stimulated groups had significant increase (P<0.05).Conclusion:(1) TGF-β1 can activate Rho/ROCK signaling pathway in HK-2 cells. (2) TGF-β1-induced Rho/ROCK signaling pathway may reduce expression of tight junctions proteins of HK-2 cells to promote EMT.