Abstract
Transforming growth factor-β (TGF-β) acts as a key cytokine in epithelial−mesenchymal transition (EMT) and myofibroblast differentiation, which are important for normal tissue repair and fibrotic diseases. Ubiquitylation and proteasomal degradation of TGF-β signaling proteins acts as a regulatory mechanism for the precise control of TGF-β signaling. SMAD-specific ubiquitin E3 ligase (SMAD ubiquitination regulatory factor 2, SMURF2) controls TGF-β signaling proteins including the TGF-β receptor (TGFR) and SMAD2/3. Here, we report that tetratricopeptide repeat domain 3 (TTC3), a ubiquitin E3 ligase, positively regulates TGF-β1-induced EMT and myofibroblast differentiation, through inducing ubiquitylation and proteasomal degradation of SMURF2. In human bronchial epithelial cells (BEAS-2B) and normal human lung fibroblasts, TTC3 knockdown suppressed TGF-β1-induced EMT and myofibroblast differentiation, respectively. Similarly, when TTC3 expression was suppressed, the TGF-β1-stimulated elevation of p-SMAD2, SMAD2, p-SMAD3, and SMAD3 were inhibited. In contrast, overexpression of TTC3 caused both EMT and myofibroblast differentiation in the absence of TGF-β1 treatment. TGF-β1 reduced SMURF2 levels and TTC3 overexpression led to a further decrease in SMURF2 levels, while TTC3 knockdown inhibited TGF-β1-induced SMURF2 reduction. In cell and in vitro ubiquitylation assays demonstrated TTC3-mediated SMURF2 ubiquitylation, and coimmunoprecipitation assays established the binding between SMURF2 and TTC3. TGF-β1-induced TTC3 expression was inhibited by the knockdown of SMAD2 and SMAD3. Finally, Ttc3 mRNA levels were significantly increased and Smurf2 protein levels were significantly decreased in the lungs of mice treated with bleomycin as compared with the lungs of control mice. Collectively, these data suggest that TTC3 may contribute to TGF-β1-induced EMT and myofibroblast differentiation, potentially through SMURF2 ubiquitylation/proteasomal degradation and subsequent inhibition of SMURF2-mediated suppression of SMAD2 and SMAD3, which in turn induces TTC3 expression.
Highlights
The epithelial−mesenchymal transition (EMT) is observed in physiological processes such as development and wound healing, and in pathological processes such as fibrotic diseases and cancer metastasis[1,2]
Given that (1) tetratricopeptide repeat domain 3 (TTC3) induced by cigarette smoke extract caused cell death, possibly through the ubiquitylation and degradation of Akt[17], (2) cigarette smoking is associated with idiopathic pulmonary fibrosis (IPF)[22,23,24], (3) Akt inhibition ameliorated pulmonary fibrosis in bleomycin-treated mice[25], and (4) Akt is one of the noncanonical signaling arms of TGF- β26 that is responsible in fibrotic changes in IPF27, we hypothesized that TTC3 might affect Transforming growth factor-β (TGF-β)-mediated EMT and myofibroblast differentiation, characteristic features of fibrotic diseases including IPF27
Considering the previous reports indicating that SMURF2 acts as a negative regulator of TGF-β signaling by modifying its related proteins including SMAD2 and SMAD311–14, we addressed whether TTC3 might cause SMURF2 reduction
Summary
The epithelial−mesenchymal transition (EMT) is observed in physiological processes such as development and wound healing, and in pathological processes such as fibrotic diseases and cancer metastasis[1,2]. Kim et al Cell Death and Disease (2019)10:92 components, together with a downregulation of epithelial signature genes including E-cadherin and zona occludens-. Similar to EMT, TGF-β potently induces myofibroblast differentiation in normal wound healing and fibrotic diseases. There is a regression and disappearance of myofibroblasts by apoptosis during normal wound healing, and the perpetual existence of myofibroblasts may be the cause of some fibrotic diseases. Resident fibroblasts and mesenchymal cells derived from epithelial cells during EMT are important sources of myofibroblasts that are involved in pathological fibrosis such as pulmonary fibrosis[7]
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