Simvastatin attenuates TGF-β1-induced epithelial-mesenchymal transition in human alveolar epithelial cells.

Transforming growth factor-β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) of alveolar epithelial cells (AEC) may contribute to idiopathic pulmonary fibrosis (IPF). TGF-β1-induced EMT in A549 cells (a human AEC cell line) resulted in the adoption of mesenchymal responses that were predominantly mediated via the TGF-β1-Smad2/3 signaling pathway. Simvastatin (Sim), a 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitor, has been previously reported to inhibit EMT in human proximal tubular epithelial cells and porcine lens epithelial cells and to suppress Smad2/3 phosphorylation in animal models. However, whether Sim can attenuate TGF-β1-induced EMT in A549 cells and its underlying mechanisms remains unknown. Cells were incubated with TGF-β1 in the presence or absence of Sim. The epithelial marker E-cadherin (E-Cad) and the mesenchymal markers, α-smooth muscle actin (α-SMA), vimentin (Vi) and fibronectin (FN), were detected using western blotting analyses and immunofluorescence. Phosphorylated Smad2 and Smad3 levels and connective tissue growth factor (CTGF) were analyzed using western blotting. In addition, a cell migration assay was performed. Moreover, the levels of matrix metalloproteinase (MMP)-2 and -9 in the culture medium were examined using ELISA. Sim significantly attenuated the TGF-β1-induced decrease in E-Cad levels and elevated the levels of α-SMA, Vi and FN via the suppression of Smad2 and Smad3 phosphorylation. Furthermore, Sim inhibited the mesenchymal-like responses in A549 cells, including cell migration, CTGF expression and secretion of MMP-2 and -9. However, Sim failed to reverse the cell morphologial changes induced by TGF-β1 in A549 cells. Sim attenuated TGF-β1-induced EMT in A549 cells and might be a promising therapeutic agent for treating IPF.

Yang T ，Chen M ，Sun T 《-》

TGF-beta1 induces human alveolar epithelial to mesenchymal cell transition (EMT).

Kasai H ，Allen JT ，Mason RM ，Kamimura T ，Zhang Z ... -  《RESPIRATORY RESEARCH》

High-Mobility Group Box 1 Mediates Epithelial-to-Mesenchymal Transition in Pulmonary Fibrosis Involving Transforming Growth Factor-β1/Smad2/3 Signaling.

Epithelial-to-mesenchymal transition (EMT) is a crucial event in the cellular origin of myofibroblasts that secrete extracellular matrix in the progression of pulmonary fibrosis (PF). High-mobility group box 1 (HMGB1) is a novel mediator of EMT. However, whether this process involves the recognized transforming growth factor-β1 (TGF-β1)/Smad signaling that also contributes to EMT in PF has not yet been elucidated. Here, we developed a model of PF induced by bleomycin (BLM) in rats and conducted several simulation experiments in A549 (human) and RLE-6TN (rat) alveolar epithelial cell (AEC) lines to unravel the role of TGF-β1/Smad2/3 signaling in HMGB1-mediated EMT. We found that the levels of serum HMGB1 and lung hydroxyproline were severely elevated after BLM administration. Moreover, the protein expression of HMGB1, TGF-β1, phosphorylated Smad2/3 (p-Smad2/3), and mesenchymal markers including α-smooth muscle actin, vimentin, and type I collagen were significantly increased with the reduced protein expression of an epithelial marker (E-cadherin) in the rat model by Western blot or immunohistochemical analysis. In addition, the uptake of both exogenous TGF-β1 and HMGB1 by AECs could induce EMT; meanwhile, HMGB1 dramatically enhanced TGF-β1 expression and triggered Smad2/3 phosphorylation. In contrast, TGF-β1 deficiency evidently ameliorated HMGB1-mediated EMT with reduced p-Smad2/3 in A549 cells. It provides new insights that HMGB1 release from injured lungs promotes AEC damage through induction of the EMT process, in which TGF-β1/Smad2/3 signaling is activated and contributes to PF. These results suggest that HMGB1 may constitute a therapeutic target for developing antifibrotic agents for abnormal lung remodeling.

Li LC ，Li DL ，Xu L ，Mo XT ，Cui WH ，Zhao P ，Zhou WC ，Gao J ，Li J ... -  《-》

Simvastatin inhibits transforming growth factor-β1-induced expression of type I collagen, CTGF, and α-SMA in keloid fibroblasts.

Simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme-A reductase inhibitor, is used to reduce cholesterol levels. Accumulating evidence has revealed the immunomodulatory and anti-inflammatory effects of simvastatin that prevent cardiovascular diseases. In addition, the beneficial effects of statins on fibrosis of various organs have been reported. However, the functional effect of statins on dermal fibrosis of keloids has not yet been explored. The objective of this study was to determine whether simvastatin could affect dermal fibrosis associated with keloids. We examined the effect of simvastatin on transforming growth factor (TGF)-β1-induced production of type I collagen, connective tissue growth factor (CTGF or CCN2), and α-smooth muscle actin (α-SMA). Keloid fibroblasts were cultured and exposed to different concentrations of simvastatin in the presence of TGF-β1, and the effects of simvastatin on TGF-β1-induced collagen and CTGF production in keloid fibroblasts were determined. The type I collagen, CTGF, and α-SMA expression levels and the Smad2 and Smad3 phosphorylation levels were assessed by Western blotting. The effect of simvastatin on cell viability was evaluated by assessing the colorimetric conversion of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. Simvastatin suppressed TGF-β1-induced type I collagen, CTGF, and α-SMA production in a concentration-dependent manner. The TGF-β1-induced Smad2 and Smad3 phosphorylation levels were abrogated by simvastatin pretreatment. The inhibition of type I collagen, CTGF, and α-SMA expression by simvastatin was reversed by geranylgeranyl pyrophosphate, suggesting that the simvastatin-induced cellular responses were due to inhibition of small GTPase Rho involvement. A RhoA activation assay showed that preincubation with simvastatin significantly blocked TGF-β1-induced RhoA activation. The Rho-associated coiled kinase inhibitor Y27632 abrogated TGF-β1-induced production of type I collagen, CTGF, and α-SMA. However, Y27632 had no significant effect on TGF-β1-induced phosphorylation of Smad2 and Smad3. In conclusion, the present study suggests that simvastatin is an effective inhibitor of TGF-β1-induced type I collagen, CTGF, and α-SMA production in keloid fibroblasts.

Mun JH ，Kim YM ，Kim BS ，Kim JH ，Kim MB ，Ko HC ... -  《-》

Astragaloside IV attenuates high glucose-induced EMT by inhibiting the TGF-β/Smad pathway in renal proximal tubular epithelial cells.

In the present study, we examined the molecular mechanism of astragaloside IV (AS-IV) in high glucose (HG)-induced epithelial-to-mesenchymal transition (EMT) in renal proximal tubular epithelial cells (PTCs). NRK-52E cell viability and apoptosis were determined by the cell counting kit-8 (CCK-8) assay and flow cytometric analysis, respectively. Expressions of E-cadherin, N-cadherin, vimentin, and occludin were measured by Western blot, and those of E-cadherin and N-cadherin were additionally measured by immunofluorescence analysis. Transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) expressions were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. The expressions of Smad2, Smad3, phosphorylated-Smad2 (p-Smad2), and p-Smad3 were measured using Western blot. We found that AS-IV could recover NRK-52E cell viability and inhibit HG-induced cell apoptosis. TGF-β1, α-SMA, Smad2, Smad3, p-Smad2, and p-Smad3 expressions were decreased in the AS-IV-treated groups compared with the HG group. Moreover, the expressions of E-cadherin and occludin were remarkably up-regulated and those of N-cadherin and vimentin were down-regulated in the AS-IV-treated groups compared with the HG group. Interestingly, the TGF-β1 activator SRI-011381 hydrochloride had an antagonistic effect to AS-IV on HG-induced EMT behavior. In conclusion, AS-IV attenuates HG-induced EMT by inhibiting the TGF-β/Smad pathway in renal PTCs.

Wang YN ，Zhao SL ，Su YY ，Feng JX ，Wang S ，Liao XM ，Wang LN ，Li JC ，Meng P ，Li HY ，Zhang YF ... -  《-》