ABSTRACT
Recently, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is suggested as a new agent in the fighting against fibrogenesis. In tumor, DJ-1 is identified as a negative regulator of PTEN. But the expression of DJ-1 and the regulation of PTEN in fibrosis are unclear. Renal fibrosis was induced in 5/6 subtotal nephrectomy rat model. Human proximal tubular epithelial cells (HKC) were treated with transforming growth factor-beta 1 (TGF-β1), or transfected with DJ-1 or PTEN. Confocal microscope was used to investigate the localization of DJ-1 and PTEN. The selective phosphoinositide-3 kinase (PI3K) inhibitor, LY294002, was administered to inhibit PI3K pathway. The DJ-1 and PTEN expression, markers of epithelial-mesenchymal transition (EMT) and Akt phosphorylation were measured by RT-PCR, Western blotting or immunocytochemistry. In vitro, after HKC cells were stimulated with 10 ng/mL TGF-β1 for 72 h, the expression of DJ-1 was increased, and that of PTEN was decreased. In vivo, the same results were identified in 5/6-nephrectomized rats. In normal HKC cells, most of DJ-1 protein localized in cytoplasm, and little in nucleus. TGF-β1 upregulated DJ-1 expression in both cytoplasma and nuclei. In contrary, TGF-β1 emptied cytoplasmic PTEN protein into nucleus. Overexpression of DJ-1 decreased the expression of PTEN, promoted the activation of Akt and the expression of vimentin, and also led to the loss of cytoplasmic PTEN. Contrarily, overexpression of PTEN protected HKC cells from TGF-β1-induced EMT. In conclusion, DJ-1 is upregulated in renal fibrosis and DJ-1 mediates EMT by suppressing cytoplasmic PTEN expression and Akt activation.
ABSTRACT
Objective To investigate the inhibitory effects of overexpression of PTEN on renal epithelial-mesenchymal trans-differentiation induced by TGF-β1,and the signaling transduction mechanism.Methods HKC cells were transfected with GFP-PTEN via lipofectAMINE2000.The efficiency of transfection was detected by fluorescence microscope.The expression of PTEN protein and mRNA in the translected cells was detected by Western blot and RT-PCR respectively.The experiment was divided into four groups:normal group,TGF-β1 stimulation group,GFP-PTEN+TGF-β1 group and empty vector+TGF-β1 group.The expression of E-cadherin,a-SMA,Akt and p-Akt was detected by Western blot.Results Most ceils transfected with GFP-PTEN expressed GFP.The expression of PTEN protein and mRNA was strongly increased when HKC cells were transfected with GFP-PTEN(all P<0.05).In both TGF-β1 stimulation group and empty vector+TGF-β1 group,the expression level of E-cadherin was lower(all P<0.05),while that of p-Akt and a-SMA was higher than in normal group(both P<0.05).The expression level of p-Akt and a-SMA in GFP-PTEN+TGF-β1 group was Iower(both P<0.05),while that of E-cadherin was higher than in TGF-β1 stimulation group and empty vector+TGF-β1 group(both P<0.05).The expression of Akt was similar in the four groups.Conclusion Overexpression of PTEN can inhibit renal epithelial-mesenehymal trans-differentiation induced by TGF-β1 through suppressing the activation of PI3K/Akt signal pathway.
ABSTRACT
Objective To observe the expression and localization of D J-1 in renal fibrosis, and to investigate the expressions of E-cadherin, vimentin and the level of β-catenin tyrosine phosphorylation in human tubular epithelial cells. Methods In vitro, the human tubular epithehal cells (HKC cell line) were cultured with 10 μg/L TGF-β1 for 72 h. The protein expressions of E-cadherin, vimentin and DJ-1 were measured by Western blot. RT-PCR was used to detect the expression of D J-1 mRNA. The intracellular distribution of DJ-1 was observed by confocal microscope. In vivo, Masson stain was used to evaluate the level of renal fibrosis. The expression and disposition of DJ-1 in renal tissue were detected by immunohistochemistry. HKC cells were transfected with pEGFP-N1-DJ-1 via lipofectamine 2000. The efficiency of transfection was detected by fluorescence microscope. The expressions of DJ-1, E-cadherin, vimentin and β-catenin tyrosine phosphorylation level in the transfected cells were detected by Western blot. Results The expressions of DJ-1 protein and DJ-1 mRNA were up-regulated in renal tubular EMT cells. Most of DJ-1 protein localized in cytoplasm, and some was in nucleus. After stimulation by TGF-β1, the expressions of DJ-1 protein both in cytoplasm and nucleus was greatly increased, especially in nucleus. In vivo, renal tissue expressed DJ-1 in tubular epithelia, but little expression in glomeruli. In renal tissue from 5/6-nephrectomized rots, DJ-1 expression was greatly increased. In the DJ-1 transfectants, the expressions of DJ-1, vimentin and β-catenin tyrosine phosphorylation level were up-regulated, but E-cadherin expression was suppressed. Conclusion The increased expression of DJ-1 may promote renal fibrosis.