Effects Of BMP-7 On Epithelial-to-Mensenchymal Transition And Extracellular Matrix Accumulation In Human Renal Proximal Tubular Epithelial Cells Induced By TGF-β₁

Objective: To investigate the effects of bone morphogenic protein(BMP)-7 on epithelial-to-mesenchymal transition(EMT) and the expression of fibronectin(FN) and type collagenⅠ(ColⅠ) in human renal proximal tubular cells(HK-2) induced by transforming growth factor-β1(TGF-β1),and to explore the possible mechanisms of BMP-7 for the inhibition and reversal of renal interstitial fibrosis.Methods: HK-2 cells were treated with TGF-β1 or a combination of TGF-β1 and BMP-7. HK-2 cells divided into the following groups according to different factors: Control group, TGF-β1(different time and concentration) group, 3ng/ml TGF-β1+ BMP-7 (different concentration) group. Morphological changes were assessed by phase contrast microscopy.The expression ofα-SMA and E-cadherin were analysed by indirect immunofluorescence ,RT-PCR and Western Blot respectively.The mRNA levels of extracelluar matrix components(ColⅠα1 and FN) and plasminogen activator inhibitior(PAI)-1 were measured by RT-PCR..The secretion of ColⅠand FN protein was quantitated by ELISA.Results: (1) TGF-β1 treatment of confluent HK-2 cells resulted in distinct morphological changes in a time and dose-dependent manner.The control cells displayed typical cobblestone morphology of epithelial cells. 3ng/ml TGF-β1 induced profound morphologic changes after 48h, with cells becoming elongated in shape, but addition of 200ng/ml BMP-7 for 48h restored the epithelial morphology of the HK-2 cells.(2)Indirect immunofluorescence staining showed that expression ofα-SMA could not be seen in control cells but upregulated and enhanced by 3ng/ml TGF-β1 after 48h.Incubation with 200ng/ml BMP-7 for 48h dramatically abrogated TGF-β1-inducedα-SMA expression. Treatment of 3ng/ml TGF-β1 resulted in distinct loss of Ecadherin staining in the plasma membrane of HK-2 cells but subsequent treatment with 200ng/ml BMP-7 largely restored the E-cadherin protein staining. (3)The expression ofα-SMA mRNA were upregulated by TGF-β1 in a dose-dependent manner maximally at the concentration of 10ng/ml TGF-β1. The 3ng/ml TGF-β1-induced levels ofα-SMA mRNA and protein were increased while E-cadherin decreased in a time-dependent manner. BMP-7 dramatically supressed TGF-β1-inducedα-SMA mRNA and protein expression in a dose-dependent manner. 400 ng/ml BMP-7 could significantly block the expression ofα-SMA mRNA and protein induced by 3ng/ml TGF-β1(P<0.01).Under the continuous influence of 3ng/ml TGF-β1,increasing doses of BMP-7(100～400ng/ml) substantially restored the expression of E-cadherin mRNA and protein (P<0.01, 400 ng/ml BMP-7+ TGF-β1VS. TGF-β1 alone). (4)3ng/ml TGF-β1 could increase the mRNA expression levels of FN and ColⅠα1 in a time-depedent manner.BMP-7 could inhibit the expression of ECM components(FN, ColⅠ) induced by TGF-β1 in a dose-depedent manner:400ng/ml BMP-7 could supress the expression of FN and ColⅠsignificantly(P<0.01). (5) 3ng/ml TGF-β1 could increase the expression of PAI-1 mRNA(P<0.01 VS. control).BMP-7 could inhibit the expression of PAI-1 mRNA induced by TGF-β1 in a dose-dependent manner.400ng/ml BMP-7 could inhibit the expression of PAI-1 mRNA significantly(P<0.01).Conclusions: (1) TGF-β1 can induce EMT and produce ECM components in association with an inhibition of E-cadherin expression, increase ofα-SMA, ColⅠand FN expression and loss of epithelial cell morphology.(2)BMP-7 can block and reverse TGF-β1-induced EMT by supressing the expression ofα-SMA and restoring the expression of E-cadherin in a dose-depedent manner.(3)BMP-7 can prevent accumulation of ECM components and enhance degradation of ECM components induced by TGF-β1 through reducing the expression of ColⅠ,FN and PAI-1.