The Role And Mechanism Of Brachyury In TGF-β1Induced EMT Of Renal Tubular Epithelial Cells

[Background]Renal interstitial fibrosis is a progressive and potentially lethal disease and a common feature of wide variety of kidney diseases. Chronic kidney disease has become a major public health problem in China. The percent of chronic kidney disease was about10.8%in China; therefore the number of patients with chronic kidney disease was around119.5million in our country. Hence, it is necessary and urgent to establish new strategies for renal disease therapies.Tubular epithelial-to-mesenchymal transition (EMT) is well-characterized whereby fully differentiated epithelial cells undergo transition to mesenchymal phenotype, reducing expression of the epithelial markers, such as E-cadherin and ZO-1and enhancing expression of the mesenchymal markers vimentin and a-smooth muscle actin. EMT display increased motility and invasiveness, and is increasingly recognized as one of the major pathways leading to the renal fibrosis after a diverse array of injuries.Brachyury, an evolutionarily conserved and T-box transcription factor, is vital for the development of embryo in all vertebrates. Recent study have revealed that Brachyury as an important factor promoted EMT involved in cancer progression and metastasis by repressed E-cadherin transcription, an effect partially mediated by Slug, leading to loss of E-cadherin-mediated cell-cell adhesion, and activation of EMT regulator might play important roles in mediating the invasion, migration, and metastatic activity of different carcinoma cells. Transforming growth factor β1(TGF-β1), one major regulator, as a well-established inducer involved in four key steps of EMT. Although the important role of TGF-β1in promoting tubular EMT is widely accepted, the mechanism by which TGF-β1induced tubular EMT process remains largely unknown. Furthermore, Latinkic BV et al found that induction of the homologue of Brachyury in Xenopus is regulated by TGF-β signals. Given the known features of Brachyury, we hypothesized that Brachyury might contribute to TGF-β1-induced renal tubular EMT.[Aims]To investigate the regulatory role of Brachyury in TGF-β1-induced EMT and the underlying mechanisms, with the aim of better elucidating the mechanisms of EMT and laying an attractive target for progression of renal fibrosis therapies.[Methods]1. HK2cells were treated with TGF-β1for various periods of time at the concentration of5ng/ml and various dose-responses, and the expressions of Brachyury were identified by WB and real-time PCR analysis;2. The pcDNA3.1-Brachyury vector or empty vector was respectively transfected into HK2cells to up-regulate its expression and the specific Brachyury siRNA and empty vector controls was transfected into HK2cells to down-regulate its expression;3. The expression of Brachyury, epithelial cell marker and mesenchymal markers were determined by IF and WB;4. The Snail siRNA or Slug siRNA and empty vector controls respectively was transfected into HK2cells to down-regulate their expressions;5. The expression of Snail, Slug, epithelial cell marker and mesenchymal markers were assessed by WB;6. Unilateral ureteral obstruction (UUO) rat model and renal biopsy samples were selected;7. The expression of Brachyury was evaluated by WB and IHC.[Results]1. qRT-PCR assay demonstrated that the induction of Brachyury in mRAN levels was increased at2h, sustained at least to24h after TGF-β1treatment, and Brachyury mRNA was induced after lng/ml TGF-β1treatment in tubular epithelial cells. WB further validated that Brachyury protein was abundance at4h after TGF-β1and sustained to at least24h. The expression of Brachyury in HK2cells was not increase with further increase in TGF-β1concentration. The dose-response of Brachyury protein expression to TGF-β1treatment, and peaking expression was observed at treatment with5ng/ml TGF-β1；2. IF assay demonstrated that overexpression of Brachyury resulted in the staining of E-cadherin and Plakoglobin decreased, meanwhile vimentin staining was dramatically induced; Western blot analysis was used to evaluate the characteristic changes of EMT in Brachyury-overexpression HK2cells. It is interesting that overexpression of Brachyury suppressed epithelial cell marker E-cadherin and Plakogolobin expression in tubular epithelial cells; meanwhile, the levels of Fibronectin and vimentin were increase;3. Transfection with Brachyury small interfering RNA (siRNA) in TGF-β1-treated HK2cell resulted in a considerable inhibition of Brachyury protein expression and the epithelial markers, mesenchymal markers expression changed;4. pcDNA3.1vector of the Brachyury and empty vector was transfected into HK2cells respectively and the expression of Snail and Slug in mRNA and protein levels were decrease compared with the control cells;5. Knock down the expression of Brachyury, Snail and Slug in Brachyury overexpression cells and the expression of E-cadherin was upregulate and vimentin was decrease in protein levels;6. Immunohistochemistry staining showed the staining of E-cadherin and vimentin were in a time-dependent manner. Western Blot analyses suggested that Brachyury was markedly induced in the fibrotic kidney in a time-dependent manner;7. The IHC results are shown that compared with the staining in control kidneys, Brachyury, Snail and Slug were numerously located in the nuclei of IgA nephropathy renal tissues, absence of or reduced E-cadherin expression was observed in Brachyury or Slug-positive tubular epithelial cells;8. Brachuyry expression in the tubules has strong association with tubulointerstitial fibrosis.[Conclusions]1. Brachyury is induced rapidly during TGF-β1-mediated EMT;2. Brachyury mediates TGF-β1-induced EMT in HK-2cells;3. Knockdown the expression of Snail and Slug prevents TGF-β1-induced EMT;4. Expression of Brachyury is induced in the fibrotic kidney.