Overexpression of smad7 inhibits the TGF-β/Smad signaling pathway and EMT in NPHP1-defective MDCK cells

Background: Nephronophthisis (NPHP) is a kind of ciliopathy. Interstitial fibrosis occurs at the early stage of the disease. TGF-beta/Smad is a key signaling pathway in regulating interstitial fibrosis and epithelial-mesenchymal transition (EMT). In this study, we explored the activation of the TGF-beta/Smad signaling pathway and EMT in NPHP1-defective MDCK cells to further understand the pathogenesis of NPHP. Methods: NPHP1-knockdown (NPHP1(KD)) MDCK cells were constructed by recombinant lentiviral short hairpin RNA, and NPHP1-knockout (NPHP1(KO)) MDCK cells were constructed by using the CRISPR/Cas9 technique. The morphology and migration ability were observed under a microscope. Western blotting was used to detect the expression of E-cadherin, beta-catenin, alpha-smooth muscle actin (alpha-SMA), fibroblast-specific protein-1(FSP1), TGF-beta 1, Smad2, Smad3, p-Smad3, Smad4 and Smad7. The localization of Smad3 was determined by immunofluorescence assay. Results: NPHP1(KD) and NPHP1(KO) MDCK cells were spindle-shaped and presented EMT-like changes. E-cadherin and beta-catenin expression decreased, while alpha-SMA and FSP1 expression increased; the TGF-beta/Smad signaling pathway was activated, Smad2, Smad3, p-Smad3 and Smad4 expression increased, Smad3 translocated to nuclear and Smad7 expression decreased compared with those in wild type MDCK cells. Overexpression of Smad7 reversed these changes to different degrees. Conclusions: Our results indicate that NPHP1 defects induce the activation of the TGF-beta/Smad signaling pathway and EMT in MDCK cells. These factors may be implicated in the pathogenesis of interstitial fibrosis in NPHP. (C) 2021 Published by Elsevier Inc.