DNMTs Are Involved in TGF-β1-Induced Epithelial-Mesenchymal Transitions in Airway Epithelial Cells

Abstract

Chronic rhinosinusitis (CRS) pathogenesis is closely related to tissue remodeling, including epithelial-mesenchymal transition (EMT). Epigenetic mechanisms play key roles in EMT. DNA methylation, mediated by DNA methyltransferases (DNMTs), is an epigenetic marker that is critical to EMT. The goal of this study was to determine whether DNMTs were involved in TGF-β1-induced EMT and elucidate the underlying mechanisms in nasal epithelial cells and air-liquid interface cultures. Global DNA methylation and DNMT activity were quantified. DNMT expression was measured using real-time PCR (qRT-PCR) in human CRS tissues. mRNA and protein levels of DNMTs, E-cadherin, vimentin, α-SMA, and fibronectin were determined using RT-PCR and Western blotting, respectively. DNMT1, DNMT3A, and DNMT3B gene expression were knocked down using siRNA transfection. MAPK phosphorylation and EMT-related transcription factor levels were determined using Western blotting. Signaling pathways were analyzed using specific inhibitors of MAPK. We demonstrated these data in primary nasal epithelial cells and air-liquid interface cultures. Global DNA methylation, DNMT activity, and DNMT expression increased in CRS tissues. DNMT expression was positively correlated with Lund-McKay CT scores. TGF-β1 dose-dependently induced DNMT expression. Further, 5-Aza inhibited TGF-β1-induced DNMT, Snail, and Slug expression related to EMT, as well as p38 and JNK phosphorylation in A549 cells and TGF-β1-induced DNMT expression and EMT in primary nasal epithelial cells and air-liquid interface cultures. TGF-β1-induced DNMT expression leads to DNA methylation and EMT via p38, JNK, Snail, and Slug signaling pathways. Inhibition of DNMT suppressed the EMT process and therefore is potentially a CRS therapeutic strategy.

Keywords: DNA methyltransferase; epithelial–mesenchymal transition; primary nasal epithelial cells; tissue remodeling; transforming growth factor β-1.

Figure 1

DNA methylation and DNMT expression in nasal tissues: (a) global DNA methylation and (b) DNMT activity were analyzed using a Global DNA Methylation Assay Kit and DNMT Activation Assay Kit in nasal mucosa tissue from healthy controls and CRS patients. Global DNA methylation and DNMT activity were significantly increased in CRSsNP and CRSwNP, compared with those in the control; (c–e) DNMT1, DNMT3A, and DNMT3B mRNA expressions were analyzed using real-time PCR in nasal mucosa tissue from healthy controls and CRS patients; (f–h) the Lund–McKay score was correlated with DNMT1, DNMT3A, and DNMT3B expression in nasal mucosa tissue. UP: uncinate process, NP: nasal polyp. Data are expressed as the mean ± SEM. * p < 0.05 vs. control.

Figure 2

TGF-β1-induced DNMT expression in A549 cells: (a) DNMT mRNA and (b) protein expression were measured using real-time PCR and Western blotting, respectively, in A549 cells after stimulation with Poly IC (10 μg/mL), LPS (10 ng/mL), TGF-β1 (5 ng/mL), IFN-γ (100 ng/mL), IL-13 (10 ng/mL), TNF-α (50 ng/mL), and IL-1β (10 ng/mL) for 24 h; (c) real-time PCR analyses of DNMT1, DNMT3A, and DNMT3B revealed dose-dependent effects in mRNA expression following TGF-β1 treatment; (d) protein expression of DNMT1, DNMT3A, and DNMT3B with TGF-β1 treatment in a dose-dependent manner was measured using Western blotting; (e) DNMT activity and (f) global DNA methylation with TGF-β1 treatment in a dose-dependent manner were detected using a Global DNA Methylation Assay Kit and DNMT Activation Assay Kit. Data are expressed as the mean ± SEM of three independent experiments using a single A549 cell line. * p < 0.05 vs. control.

Figure 3

Effect of 5-Aza on EMT induced by TGF-β1 in A549 cells. A549 cells were stimulated with TGF-β1 (5 ng/mL) with or without the DNMT inhibitor (5-Aza, 10 μM): (a) the mRNA expression levels of DNMT1, DNMT3A, and DNMT3B were determined using RT–PCR; (b) protein expression levels of DNMT1, DNMT3A, and DNMT3B were measured using Western blotting; (c,d) effects of 5-Aza on the mRNA and protein levels of E-cadherin, vimentin, α-smooth muscle actin protein, and fibronectin expression in TGF-β1-stimulated A549 cells as determined using RT–PCR and Western blotting. Data are expressed as the mean ± SEM of three independent experiments. Values are expressed as the mean ± SEM of three independent samples using a single A549 cell line. * p < 0.05 vs. control; † p < 0.05 vs. TGF-β1 alone.

Figure 4

TGF-β1-induced EMT in A549 cells was inhibited by DNMT1, DNMT3A, and DNMT3B siRNAs. Specific DNMT1, DNMT3A, and DNMT3B siRNAs were transfected before treatment with or without TGF-β1 for 24 h for mRNA expression and 72 h for protein expression in A549 cells: (a–c) mRNA levels of DNMT1, DNMT3A, and DNMT3B were determined using real-time PCR; (d–f) protein levels of DNMT1, DNMT3A, and DNMT3B were determined using Western blotting; (g–i) mRNA levels of EMT-related markers (E-cadherin, vimentin, α-smooth muscle actin protein, and fibronectin) were measured using real-time PCR after transfection of DNMT1, DNMT3A, or DNMT3B siRNA with or without TGF-β1 for 24 h; (j–l) Protein levels of EMT-related markers were measured using Western blotting after transfection of DNMT1, DNMT3A, or DNMT3B siRNA with or without TGF-β1 for 72 h. Data are expressed as the mean ± SEM of three independent experiments using a single A549 cell line. * p < 0.05 vs. control with siControl; † p < 0.05 vs. TGF-β1 with siControl.

Figure 5

Signaling pathways of TGF-β1 on DNMT expression. A549 cells were pretreated with ERK inhibitor (10 µM U0126), p38 inhibitor (10 µM SB203580), JNK inhibitor (10 µM SP600125), and 5-Aza for 1  h, and then stimulated with TGF-β1: (a) mRNA and (b) protein expression levels were determined using real-time PCR and Western blotting; (c) DNMT activity and (d) global DNA methylation were detected using the Global DNA Methylation Assay Kit and DNMT Activation Assay Kit, respectively. Data are expressed as the mean ± SEM of three independent experiments using a single A549 cell line. * p < 0.05 vs. control; † p < 0.05 vs. TGF-β1 alone.

Figure 6

EMT-related transcription factor with TGF-β1 treatment on DNMT expression. A549 cells were pretreated with 5-Aza before TGF-β1 treatment: (a) mRNA and (b) protein levels of Snail and Slug were determined using real-time PCR and Western blotting, respectively, normalized to GAPDH. Specific Snail and Slug siRNAs (100 nM) were transiently transfected before treatment with or without TGF-β1 (5 ng/mL) for 24 and 72 h; (c–j) expression of DNMT and EMT-related markers was determined using real-time PCR and Western blotting. Data are presented as mean  ±  SEM. Results were obtained from at least three independent experiments using a single A549 cell line. * p  <  0.05 vs. control; † p  <  0.05 vs. TGF-β1 with siControl.

Figure 7

Effects of 5-Aza on EMT induced by TGF-β1 in primary nasal epithelial cells and air–liquid interface culture. Human primary nasal epithelial cells were stimulated with TGF-β1 (5 ng/mL) with or without the DNMT inhibitor (5-Aza, 10 μM): (a,b) mRNA expression levels of DNMT1, DNMT3A, DNMT3B, and EMT-related markers were determined using RT–PCR; (c) protein expression levels of DNMT1, DNMT3A, DNMT3B, and EMT-related markers were measured using Western blotting; (d) effects of 5-Aza on E-cadherin, DNMT1A, DNMT3A, and DNMT3B expression in TGF-β1-stimulated human primary nasal epithelial cells were determined using immunofluorescence. Values are expressed as the mean ± SEM of three independent samples using primary nasal epithelial cells isolated from three different CRS patients. * p < 0.05 vs. control; † p < 0.05 vs. TGF-β1 alone.