title: TGF-β1 induces EMT reprogramming of porcine bladder urothelial cells into collagen producing fibroblasts-like cells in a Smad2/Smad3-dependent manner.
abstract: Activation of fibroblasts and their differentiation into myofibroblasts, excessive collagen production and fibrosis occurs in a number of bladder diseases. Similarly, conversion of epithelial cells into mesenchymal cells (EMT) has been shown to increase fibroblasts like cells. TGF-β1 can induce the EMT and the role of TGF-β1-induced EMT during bladder injury leading to fibrosis and possible organ failure is gaining increasing interest. Here we show that EMT and fibrosis in porcine bladder urothelial (UC) cells are Smad dependent. Fresh normal porcine bladder urothelial cells were grown in culture with or without TGF-β1 and EMT markers were assessed. TGF-β1 treatment induced changes in cellular morphology as depicted by a significant decrease in the expression of E-cadherin and corresponding increase in N-cadherin and α-SMA. We knocked down Smad2 and Smad3 by Smad specific siRNA. Downregulation of E-cadherin expression by TGF-β1 was Smad3-dependent, whereas N-cadherin and α-SMA were dependent on both Smad2 and Smad3. Connective tissue growth factor (CTGF/CCN2), matrix metalloproteinase-2 and -9 (MMP-2, MMP-9) has been shown to play important roles in the pathogenesis of fibrosis. Induction of these genes by TGF-β1 was found to be time dependent. Upregulation of CTGF/CCN2 by TGF-β1 was Smad3 dependent; whereas MMP-2 was Smad2 dependent. Smad2 and Smad3 both participated in MMP-9 expression. TGF-β1 reprogrammed mesenchymal fibroblast like cells robustly expressed collagen I and III and these was inhibited by SB-431542, a TGF-β receptor inhibitor. Our results indicate that EMT of porcine bladder UC cells is TGF-β1 dependent and is mediated through Smad2 and Smad3. TGF-β1 may be an important factor in the development of bladder fibrosis via an EMT mechanism. This identifies a potential amenable therapeutic target.
authors: Program in Developmental & Stem Cell Biology, The Hospital for Sick Children Research Institute, Toronto, ON, Canada, syed.islam@sickkids.ca.
authors: Syed S
authors: SS
authors: Islam
authors: Reza Bayat
authors: RB
authors: Mokhtari
authors: Yaser
authors: Y
authors: El Hout
authors: M A
authors: MA
authors: Azadi
authors: M
authors: M
authors: Alauddin
authors: Herman
authors: H
authors: Yeger
authors: Walid A
authors: WA
authors: Farhat