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

Abstract

Background: Fibroblastic foci are characteristic features in lung parenchyma of patients with idiopathic pulmonary fibrosis (IPF). They comprise aggregates of mesenchymal cells which underlie sites of unresolved epithelial injury and are associated with progression of fibrosis. However, the cellular origins of these mesenchymal phenotypes remain unclear. We examined whether the potent fibrogenic cytokine TGF-beta1 could induce epithelial mesenchymal transition (EMT) in the human alveolar epithelial cell line, A549, and investigated the signaling pathway of TGF-beta1-mediated EMT.

Methods: A549 cells were examined for evidence of EMT after treatment with TGF-beta1. EMT was assessed by: morphology under phase-contrast microscopy; Western analysis of cell lysates for expression of mesenchymal phenotypic markers including fibronectin EDA (Fn-EDA), and expression of epithelial phenotypic markers including E-cadherin (E-cad). Markers of fibrogenesis, including collagens and connective tissue growth factor (CTGF) were also evaluated by measuring mRNA level using RT-PCR, and protein by immunofluorescence or Western blotting. Signaling pathways for EMT were characterized by Western analysis of cell lysates using monoclonal antibodies to detect phosphorylated Erk1/2 and Smad2 after TGF-beta1 treatment in the presence or absence of MEK inhibitors. The role of Smad2 in TGF-beta1-mediated EMT was investigated using siRNA.

Results: The data showed that TGF-beta1, but not TNF-alpha or IL-1beta, induced A549 cells with an alveolar epithelial type II cell phenotype to undergo EMT in a time-and concentration-dependent manner. The process of EMT was accompanied by morphological alteration and expression of the fibroblast phenotypic markers Fn-EDA and vimentin, concomitant with a downregulation of the epithelial phenotype marker E-cad. Furthermore, cells that had undergone EMT showed enhanced expression of markers of fibrogenesis including collagens type I and III and CTGF. MMP-2 expression was also evidenced. TGF-beta1-induced EMT occurred through phosphorylation of Smad2 and was inhibited by Smad2 gene silencing; MEK inhibitors failed to attenuate either EMT-associated Smad2 phosphorylation or the observed phenotypic changes.

Conclusion: Our study shows that TGF-beta1 induces A549 alveolar epithelial cells to undergo EMT via Smad2 activation. Our data support the concept of EMT in lung epithelial cells, and suggest the need for further studies to investigate the phenomenon.

Figure 1

Expression changes of EMT-related markers in A549 cells. (A) A549 cells were incubated with up to 10 ng/ml of TGF-β1 in the absence of serum for up to 72 h. Expression of the epithelial marker E-cadherin is down-regulated by TGF-β1 stimulation in a concentration-and time-dependent manner. Expression of Fn-EDA, which is a mesenchymal marker, is up-regulated by TGF-β1 in parallel with the down regulation in the epithelial marker. The same amounts of total protein are loaded in each lane. (B) Densitometric analysis of band intensities for each EMT related marker was performed at 48 h. Each bar represents mean ± SD of three independent experiments. * P < 0.05 and ** P < 0.01.

Figure 2

Morphological changes induced by TGF-β1. A549 cells were incubated with 5 ng/ml of TGF-β1 for 48 h. (A) Untreated A549 cells show a pebble-like shape and cell-cell adhesion is clearly observed. (B) TGF-β1-treated cells show a decrease in cell-cell contacts and adopt a more elongated morphological shape (magnification of 200×).

Figure 3

Comparison of EMT-related marker expression in response to TGF-β1, IL-1β and TNF-α treatments. A549 cells were incubated with TGF-β1, IL-1β and TNF-α at the indicated concentrations for 48 h. Only TGF-β1 decreases E-cadherin expression concomitant with increasing Fn-EDA expression. In contrast, TNF-α only slightly decreases E-cadherin expression and IL-1β has no influence on EMT-related marker expression. Equal amounts of total protein are loaded in each lane.

Figure 4

TGF-β1 induces the expression of collagens type I and type III. A549 cells were incubated with 5 ng/ml of TGF-β1 in the presence of 5 μg/ml of L-ascorbic acid for 72 h. (A) mRNA expression of collagens type I and type III was detected using RT-PCR. Densitometric analysis was performed. The changes of expression level are expressed as fold increase compared to the control. Each bar represents the mean ± SD of three independent experiments. ** P < 0.01. (B) Protein expression of collagens type I and type III was detected by immunocytochemical staining. Panels (a) and (b) represent collagen type I and panels (c) and (d) represent collagen type III expression, respectively. TGF-β1 induces collagen type I and type III expression in A549 as shown in panel (b) and (d).

Figure 5

TGF-β1 increases collagen type IV secretion in A549. A549 cells were incubated with several concentrations of TGF-β1 in the presence of 5 μg/ml of L-ascorbic acid for 72 h. Concentrations of collagen type IV in conditioned media were determined using ELISA. TGF-β1 increases collagen type IV in a concentration-dependent manner. Each bar was expressed as the mean ± SD of four independent experiments. ** P < 0.01.

Figure 6

TGF-β1 induces CTGF expression in A549. A549 cells were incubated for the times indicated with the concentrations of TGF-β1 shown. Cell lysates were used as a source for Western blot analysis of CTGF expression. Up-regulation of CTGF expression is observed with 1 ng/ml and higher concentrations of TGF-β1. This phenomenon parallels the altered expression of EMT related markers. Representative blots are shown from three independent experiments.

Figure 7

Effect of TGF-β1 on MMPs expression in A549. Gelatin zymography was performed using the conditioned media that were harvested after 48 h TGF-β1 treatment (0.1 to 5 ng/ml). The samples were applied without reduction to a 10% polyacrylamide gel containing gelatin, and proteolytic activity was demonstrated by digestion of the gelatin and clearing of the gel.

Figure 8

Activation of Smad and ERK1/2 pathways by TGF-β1. A549 cells were pre-incubated in the presence or the absence of 10 μM of U0126, a potent MEK inhibitor, for 1 h prior to TGF-β1 stimulation. 5 ng/ml of TGF-β1 was used as a stimulus. TGF-β1 activates Erk1/2 and Smad2 pathways within 5 min after stimulation. The MEK inhibitor blocks Erk1/2 phosphorylation, but does not influence Smad2 phosphorylation. Equal amounts of total protein are loaded in each lane.

Figure 9

Effect of Smad2 siRNA on TGF-β1 induced EMT in A549 cells. Pooled synthetic siRNA duplexes targeting different regions of Smad2 were transfected into A549 cells at 50 pmol per well. 24 h after transfection, cells were stimulated with 5 ng/ml of TGF-β1 in serum free 0.1% BSA/DMEM for a further 48 h prior to harvest. Equal amounts of lysates were resolved by SDS-PAGE and analyzed by Western blotting for expression of proteins.