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Melatonin Inhibits Transforming Growth Factor-β1-Induced Epithelial-Mesenchymal Transition in AML12 Hepatocytes

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Melatonin Inhibits Transforming Growth Factor-β1-Induced Epithelial-Mesenchymal Transition in AML12 Hepatocytes

Jung-Yeon Kim et al. Biology (Basel).

Abstract

Recent studies showed that melatonin, a well-known pineal hormone that modulates the circadian rhythm, exerts beneficial effects against liver fibrosis. However, mechanisms for its protective action against the fibrotic processes remain incompletely understood. Here, we aimed to explore the effects of the hormone on transforming growth factor-β1 (TGF-β1)-stimulated epithelial-mesenchymal transition (EMT) in AML12 hepatocytes. Pretreatment with melatonin dose-dependently reversed downregulation of an epithelial marker and upregulation of mesenchymal markers after TGF-β1 stimulation. Additionally, melatonin dose-dependently suppressed an increased phosphorylation of Smad2/3 after TGF-β1 treatment. Besides the canonical Smad signaling pathway, an increase in phosphorylation of extracellular signal-regulated kinase 1/2 and p38 was also dose-dependently attenuated by melatonin. The suppressive effect of the hormone on EMT stimulated by TGF-β1 was not affected by luzindole, an antagonist of melatonin membrane receptors, suggesting that its membrane receptors are not required for the inhibitory action of melatonin. Moreover, melatonin suppressed elevation of intracellular reactive oxygen species (ROS) levels in TGF-β1-treated cells. Finally, TGF-β1-stimulated EMT was also inhibited by the antioxidant N-acetylcysteine. Collectively, these results suggest that melatonin prevents TGF-β1-stimulated EMT through suppression of Smad and mitogen-activated protein kinase signaling cascades by deactivating ROS-dependent mechanisms in a membrane receptor-independent manner.

Keywords: epithelial-mesenchymal transition; liver fibrosis; melatonin; reactive oxygen species; transforming growth factor-β1.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Figures

Figure 1
Figure 1
Effects of melatonin on mRNA expression of epithelial–mesenchymal transition (EMT) markers in transforming growth factor-β1 (TGF-β1)-treated hepatocytes. AML12 hepatocytes were preincubated with melatonin (Mel; 0.1 mM or 1 mM) or vehicle (Veh; 0.1% dimethyl sulfoxide) for 30 min and then treated with TGF-β1 (2 ng/mL) for 48 h. Relative mRNA levels of E-cadherin (A), α-smooth muscle actin (α-SMA) (B), vimentin (C), and fibronectin (D). All data are presented as the mean ± standard error of the mean (SEM). ** p < 0.01 vs. vehicle-treated cells (Veh). # p < 0.05 vs. cells treated with TGF-β1 alone.
Figure 2
Figure 2
Effects of melatonin on protein levels of EMT markers in TGF-β1-treated hepatocytes. AML12 hepatocytes were preincubated with melatonin (Mel; 0.1 mM or 1 mM) or vehicle (Veh; 0.1% dimethyl sulfoxide) for 30 min and then treated with TGF-β1 (2 ng/mL) for 48 h. (A) Western blot image of the expression of E-cadherin, α-SMA, vimentin, fibronectin, and β-actin. The graphs show densitometric quantification of E-cadherin (B), α-SMA (C), vimentin (D), and fibronectin (E) normalized against β-actin. All data are presented as the mean ± SEM. ** p < 0.01 and *** p < 0.001 vs. vehicle-treated cells (Veh). # p < 0.05, ## p < 0.01, and ### p < 0.001 vs. cells treated with TGF-β1 alone.
Figure 3
Figure 3
Effects of melatonin on the Smad signaling pathway in TGF-β1-treated hepatocytes. AML12 hepatocytes were preincubated with melatonin (Mel; 0.1 mM or 1 mM) or vehicle (Veh; 0.1% dimethyl sulfoxide) for 30 min and then treated with TGF-β1 (2 ng/mL) for 24 h. (A) Western blot image of the expression of p-Smad2/3, Smad2/3, and β-actin. (B) Densitometric quantification of p-Smad2/3 normalized against Smad2/3. All data are presented as the mean ± SEM. *** p < 0.001 vs. vehicle-treated cells (Veh). # p < 0.05 and ### p < 0.001 vs. cells treated with TGF-β1 alone.
Figure 4
Figure 4
Effects of melatonin on the mitogen-activated protein kinase (MAPK) signaling pathway in TGF-β1-treated hepatocytes. AML12 hepatocytes were preincubated with melatonin (Mel; 0.1 mM or 1 mM) or vehicle (Veh; 0.1% dimethyl sulfoxide) for 30 min and then treated with TGF-β1 (2 ng/mL) for 24 h. (A) Western blot image of the expression of p-extracellular signal-regulated kinase 1/2 (p-ERK1/2), ERK1/2, p-p38, p38, p-c-Jun N-terminal kinase 1/2 (p-JNK1/2), JNK1/2, and β-actin. The graphs show densitometric quantification of p-ERK1/2 (B), p-p38 (C), and p-JNK1/2 (D) normalized against the total level of each protein. All data are presented as the mean ± SEM. * p < 0.05, ** p < 0.05, and *** p < 0.001 vs. vehicle-treated cells (Veh). # p < 0.05 and ## p < 0.01 vs. cells treated with TGF-β1 alone. NS: not significant.
Figure 5
Figure 5
Effects of luzindole on mRNA expression of EMT markers in TGF-β1-treated hepatocytes preincubated with melatonin. AML12 hepatocytes were preincubated with melatonin (Mel; 1 mM) or vehicle (Veh; 0.6% dimethyl sulfoxide) for 30 min in the presence or absence of luzindole (Luz; 100 μM) and then treated with TGF-β1 (2 ng/mL) for 48 h. Relative mRNA levels of E-cadherin (A), α-SMA (B), vimentin (C), and fibronectin (D). All data are presented as the mean ± SEM. ** p < 0.01 vs. vehicle-treated cells (Veh). # p < 0.05 vs. cells treated with TGF-β1 alone. NS: not significant.
Figure 6
Figure 6
Effects of luzindole on protein levels of EMT markers in TGF-β1-treated hepatocytes preincubated with melatonin. AML12 hepatocytes were preincubated with melatonin (Mel; 1 mM) or vehicle (Veh; 0.6% dimethyl sulfoxide) for 30 min in the presence or absence of luzindole (Luz; 100 μM) and then treated with TGF-β1 (2 ng/mL) for 48 h. (A) Western blot image of the expression of E-cadherin, α-SMA, vimentin, fibronectin, and β-actin. The graphs show densitometric quantification of E-cadherin (B), α-SMA (C), vimentin (D), and fibronectin (E) normalized against β-actin. All data are presented as the mean ± SEM. ** p < 0.01 and *** p < 0.001 vs. vehicle-treated cells (Veh). # p < 0.05, ## p < 0.01, and ### p < 0.001 vs. cells treated with TGF-β1 alone. NS: not significant.
Figure 7
Figure 7
Effects of N-acetylcysteine (NAC) on reactive oxygen species (ROS) generation and mRNA expression of EMT markers in TGF-β1-treated hepatocytes. (A) AML12 hepatocytes were preincubated with melatonin (Mel; 1mM), NAC (10 mM), or vehicle (Veh; 0.1% dimethyl sulfoxide) for 30 min and then treated with TGF-β1 (2 ng/mL) for 48 h. Intracellular ROS was measured using the 2′,7′-dichlorodihydrofluorescein diacetate assay. *** p < 0.001 vs. vehicle-treated cells (Veh). # p < 0.05 vs. cells treated with TGF-β1 alone. (BE) AML12 hepatocytes were preincubated with NAC (10 mM) for 30 min and then treated with 2 ng/mL TGF-β1 for 48 h. Relative mRNA levels of E-cadherin (B), α-SMA (C), vimentin (D), and fibronectin (E). All data are presented as the mean ± SEM. ** p < 0.01 and *** p < 0.001 vs. non-treated cells. # p < 0.05 and ## p < 0.01 vs. cells treated with TGF-β1 alone.
Figure 8
Figure 8
Effects of NAC on protein levels of EMT markers in TGF-β1-treated hepatocytes. AML12 hepatocytes were preincubated with NAC (10 mM) for 30 min and then treated with TGF-β1 (2 ng/mL) for 48 h. (A) Western blot image of the expression of E-cadherin, α-SMA, vimentin, fibronectin, and β-actin. The graphs show densitometric quantification of E-cadherin (B), α-SMA (C), vimentin (D), and fibronectin (E) normalized against β-actin. All data are presented as the mean ± SEM. *** p < 0.001 vs. non-treated cells. ## p < 0.01 and ### p < 0.001 vs. cells treated with TGF-β1 alone.

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