Promotion of USP4 to TGF-β1-induced EMT in renal tubular epithelial cells is regulated by Akt through stabilizing TβRI

• Published in: bioRxiv
• Main Authors: Jin-Yun, Pu, Li-Xia, Wang, Wang, Jie, Zhang, Yu, Zhou, Jian-Hua
• Format: Paper
• Language: English
• Published: Cold Spring Harbor Cold Spring Harbor Laboratory Press 28.10.2020
• Subjects: 1-Phosphatidylinositol 3-kinase; AKT protein; Cytoplasm; E-cadherin; Epithelial cells; Fibrosis; Immunohistochemistry; Kidneys; Mesenchyme; Peptidase; Phosphorylation; Protein expression; siRNA; Transforming growth factor-b1; Ubiquitin
• DOI: 10.1101/2020.10.28.358796

Abstract Objective We aimed to explore the role of ubiquitin-specific peptidase-4 (USP4) in TGF-β1 induced epithelial-mesenchymal transition (EMT) during renal fibrosis, and investigated that if Akt inactivation exerted a critical effect on EMT via USP4/TβRI pathway. Methods USP4, pAkt and TβRI proteins in the obstructed kidneys from unilateral ureteral obstruction (UUO) rats were detected by immunohistochemistry assay or western blot method. E-cadherin, α-SMA, USP4 and pAkt protein expression in NRK-52E cells at different concentration of TGF-β1 were detected at different time. Further, NRK-52E cells were transfected with USP4-specific siRNA (si-USP4), and then stimulated with 10 ng/ml TGF-β1 for 24h to detect E-cadherin, α-SMA, E-cadherin and TβRI by immunofluorescent double staining assay. Pretreated with PI3K inhibitor LY294002, protein expression levels of pAkt, E-cadherin, α-SMA were detected. Meanwhile, the location of USP4 was visualized by immunofluorescent assay in NRK-52E cells. Results The expression of USP4 and TβRI was significantly upregulated in the tubular epithelial cells of UUO rats. We also found that TGF-β1 upregulated USP4 and activated Akt in NRK-52E cells during EMT. In vitro, downregulation of USP4 inhibited TβRI expression and partially reversed EMT stimulated by TGF-β1. In the meantime, blunted phosphorylation of Akt with LY294002 promoted the E-cadherin expression, and inhibited α-SMA expression in response to TGF-β1. However, inactivation of Akt could reverse EMT process, but failed to induce USP4 to shuttle between the nucleus and the cytoplasm in NRK-52E cells stimulated by TGF-β1. Conclusions These data implied that USP4 was a harmful molecule induced by TGF-β1, regulated by Akt activation and promoted TGF-β1-induced EMT via TβRI in tubular epithelial cells during renal fibrosis.