Trichostatin A Inhibits Epithelial Mesenchymal Transition Induced by TGF-β1 in Airway Epithelium

Abstract

Background and objectives: Tissue remodeling is believed to cause recalcitrant chronic rhinosinusitis (CRS). Epithelial-mesenchymal transition (EMT) is a novel clinical therapeutic target in many chronic airway diseases related with tissue remodeling. The aim of this study was to investigate the effect of trichostatin A (TSA) on transforming growth factor (TGF)-β1-induced EMT in airway epithelium and nasal tissue.

Materials and methods: A549 cells, primary nasal epithelial cells (PNECs), or inferior nasal turbinate organ culture were exposed to TSA prior to stimulation with TGF-β1. Expression levels of E-cadherin, vimentin, fibronectin, α-smooth muscle actin (SMA), histone deacetylase 2 (HDAC2), and HDAC4 were determined by western blotting and/or immunofluorescent staining. Hyperacetylation of histone H2 and H4 by TSA was measured by western blotting. After siHDAC transfection, the effects of HDAC2 and HDAC4 silencing on expression of E-cadherin, vimentin, fibronectin, α-SMA, HDAC2, and HDAC4 in TGF-β1-induced A549 were determined by RT-PCR and/or western blotting. We assessed the change in migration capacity of A549 cells by using cell migration assay and transwell invasion assay.

Results: TGF-β1 altered mRNA and protein expression levels of EMT markers including E-cadherin, vimentin, fibronectin, α-SMA, slug, and snail in A549 cells. Inhibition and silencing of HDAC2 and HDAC4 by TSA and siRNA enhanced TGF-β1-induced EMT in A549 cells. TSA blocked the effect of TGF-β1 on the migratory ability of A549 cells. In experiments using PNECs and inferior turbinate organ cultures, TSA suppressed expression of EMT markers induced by TGF-β1.

Conclusions: We showed that EMT is induced by TGF-β1 in airway epithelial cells and nasal tissue via activation of HDAC2 and HDAC4, and that inhibition of HDAC2 and HDAC4 by TSA reduces TGF-β1-induced EMT. This observation indicates that histone deacetylase inhibitors such as TSA could be potential candidates for treatment of recalcitrant CRS related with tissue remodeling.

Fig 1. Cytotoxicity of histamine determined by MTT assay.

MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide, *P < 0.05 vs. control.

Fig 2

(A) Effects of trichostatin A on morphology of TGF-β1-stimulated A549 cells as observed under a phase contrast microscope. Effects of trichostatin A on expression of E-cadherin, vimentin, fibronectin, and α-smooth muscle actin protein in TGF-β1-stimulated A549 cells were determined by western blotting (B) and immunofluorescent staining (C). Representative of independent experiments. Scale bar = 50 μm.

Fig 3. Effects of trichostatin A on expression of snail and slug mRNA and protein in TGF-β1-stimulated A549 cells were determined by RT-PCR (A) and western blotting (B) (Representative of independent experiments).

Values are expressed as the mean ± standard error of the mean (SEM) of independent experiments. *P < 0.05 vs. control. †P < 0.05 vs. TGF-β1 alone. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig 4

Effects of trichostatin A on expression of HDAC2 and HDAC4 mRNA and protein in TGF-β1-stimulated A549 cells were determined by RT-PCR (A) and western blotting (B) (representative of independent experiments). Effects of trichostatin A on hyperacetylation of histone H3 and H4 were determined by western blotting (C) (representative of independent experiments). Values are expressed as the mean ± SEM of independent experiments. *P < 0.05 vs. control. †P < 0.05 vs. TGF-β1 alone. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig 5

Effects of siHDACs on expression of HDAC2 and HDAC4 mRNA and protein in TGF-β1-stimulated A549 cells were determined by RT-PCR (A, D) and western blotting (B, E) (representative of independent experiments). Effects of siHDACs on expression of E-cadherin, vimentin, fibronectin, and α-smooth muscle actin protein in A549 cells were determined by western blotting (C, F) (representative of independent experiments). Values are expressed as the mean ± SEM of independent experiments. *P < 0.05 vs. control. †P < 0.05 vs. TGF-β1 alone. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig 6. Effects of trichostatin A on migration ability of TGF-β1-stimulated A549 cells were measured using cell migration assay (A) and transwell invasion assay (B).

Values are expressed as the mean ± SEM of independent experiments. *P < 0.05 vs. control. †P < 0.05 vs. TGF-β1 alone. Scale bar = 50 μm.

Fig 7

Effects of trichostatin A on expression of E-cadherin, vimentin, fibronectin, α-smooth muscle actin, snail, and slug proteins in TGF-β1-stimulated primary nasal epithelial cells were determined by immunofluorescent staining (A). Effects of trichostatin A on expression of E-cadherin, vimentin, fibronectin, and α-smooth muscle actin protein in TGF-β1-stimulated inferior turbinate tissue were determined by western blotting (B). Representative of independent experiments. Scale bar = 50 μm.