Epithelial-mesenchymal transition (EMT) is the trans-differentiation of epithelial cells into mesenchymal cells. During this process, epithelial cells are removed of cellular polarity and epithelial cell-cell, as well as cell-matrix adhesion contacts are remodelled. Markers of polarized epithelial cells, such as E-cadherin and some cytokeratins, are lost whereas markers of mesenchymal cells such as vimentin, N-cadherin or of myofibroblasts, as α-smooth muscle actin (α-SMA) are acquired [1]. As a result of these changes, epithelial cells are converted into motile fibroblasts to myofibroblasts (FMT) that are key cells in both the degradation and de novo synthesis of the extracellular matrix (ECM). The trans-differentiated cells are able to migrate through basement membranes into other areas in the tissue. EMT has been shown to play a role in organogenesis during embryonic development and is an important biological process in normal wound healing [2]. However, disregulated EMT also appears to occur in disease, contributing to cancer progression and metastasis, as well as to the pathogenesis of chronic degenerative fibrotic disorders in several organs including the lung [3, 4].

Dysfunction of the lungs and conducting airways at different levels may lead to a number of respiratory diseases such as asthma, cystic fibrosis, chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis and an important role for EMT has been proposed in the pathogenesis of many of these [5–8]. Particularly, recent studies have identified important mechanisms that control the mesenchymal trans-differentiation of lung alveolar epithelial cells (AECs) in pulmonary fibrosis. It has been shown that both rat and human lung AECs can undergo EMT in response to exposure to transforming growth factor (TGF)-β1 in culture [9–11]. The EMT of alveolar cells was later demonstrated in vivo by generating transgenic mice expressing β-galactosidase exclusively in lung epithelial cells and following their fate after inducing lung fibrosis by intranasal instillation of adenoviral TGF-β1 [12]. Further evidence for the re-occurence of EMT in adult tissue, as well as for its possible role in pathogenesis, was obtained from lung tissue from idiopathic pulmonary fibrosis (IPF) patients by co-localization of myofibroblast and epithelial markers in AECs overlying fibroblastic foci, which are key features of the disease and are important for both diagnosis and disease progression [11].

A number of growth factors and cytokines are able to induce EMT in vitro. TGF-β is a central regulator of pulmonary inflammation and fibrosis [13]. TGF-β1 has been reported to induce EMT in AECs as well as the differentiation of fibroblasts to myofibroblasts and was also shown to be upregulated in IPF patients [11, 14–16]. Several other factors have also been identified that appear to act in concert with or to modulate TGF-β activity. Whereas on its own IL (interleukin)1β does not induce EMT in A549 cells and similarly, tumour necrosis factor (TNF)-α has only a mild effect, when combined with TGF-β the two factors enhance EMT in type II AECs in both rat-derived cells and in the human A549 cell line. Similarly, connective tissue growth factor (CTGF) is produced by fibroblasts and bronchial epithelial cells and is also known to regulate fibroblast proliferation and collagen deposition in coordination with TGF-β [17, 18].

The molecular mechanisms underlying EMT in the lung have not been explored in great detail. TGF-β1 is known to signal through two main pathways: the canonical Smad-dependent pathway and the alternative mitogen-activated protein kinase (MAPK) pathway [19]. Both of these have been implicated in EMT and FMT [7, 20–22]. In addition, bone morphogenetic proteins (BMPs), members of the TGF-β superfamily, are involved in a variety of biological functions including lung morphogenesis [23]. The balance and interplay between BMP and TGF-β signalling has been demonstrated to be important to development [24]. Smad2 and Smad3 are activated by activin receptor-like kinase (ALK)-4, -5 and -7 receptors in response to TGF-β ligands, whereas Smad1, Smad5 and Smad8 are activated by ALK-1, -2, -3 and -6 in response to BMPs [25]. Several BMPs, particularly BMP7 and BMP4, have been implicated in EMT in various organs such as the kidney and lung [26, 27].

The ECM also appears to be playing key roles in fibrosis not only as a major cause of the disruption of tissue architecture but also due to its ability to activate cellular pathways. The main ECM components of lung basement membranes are collagen IV and laminin [28]. For example, in IPF lung basement membranes are disrupted and the expression of collagen I, collagen III and fibronectin is upregulated [29–31]. Furthermore, fibronectin has been recently shown to drive the EMT of mouse AECII [12]. Two main families of receptors for ECM proteins have been implicated in lung cancer and fibrosis [32]: integrins and the discoidin domain receptors (DDRs). While integrins can sense various ECM proteins, DDRs appear to play a more specialized role. They are receptor tyrosine kinases that function as collagen receptors and have intrinsic signalling properties [33]. DDR1 is expressed primarily in epithelial tissues, whereas the other member of the subfamily, DDR2, is mostly present in mesenchymal cells [34]. The differential expression of DDR receptors appears to be context and disease dependent. Human nasopharyngeal carcinoma and HAK-1B hepatoma cells showed overexpression of DDR2, whereas DDR1 expression was unaltered [35, 36]. In contrast, in human lung cancer tissue DDR1 was upregulated, whereas DDR2 expression was unaffected [37]. As collagen receptors, DDRs may play a role in mechanisms related to excessive collagen deposition, such as EMT or FMT in fibrosis. However, they have not yet been explored in this context.

Another mechanism that is proposed to contribute to the remodelling of diseases is the disturbed physiological balance between matrix formation and degradation. This equilibrium appears to be lost in fibrotic diseases and several matrix metalloproteases (MMPs), involved in both matrix turnover and growth factor activation, such as MMP-2 and MMP-9, have been shown to be upregulated [38]. These two MMPs also play a role in the cleavage and activation of the main profibrotic growth factor, TGF-β, and, hence, they may be of particular importance [39].

The identification of the specific epithelial cell types that are able to undergo EMT, and the underlying mechanisms, has recently been a matter of intense investigation as this may be an important process by which fibroblast numbers increase, constituting the fibroblastic foci correlated with severity of pulmonary fibrosis [40]. As described above, most of the work on lung EMT has focused on the alveolar epithelium. The increased expression of the mesenchymal marker S100A4 in airway epithelium biopsy samples from lung transplant patients suggests that the epithelium lining the conducting airways might also be able to differentiate into mesenchymal cells [41]. In the current study, we asked whether primary human bronchial epithelial cells, similarly to AECs, can also undergo EMT. We demonstrate that these cells can phenotypically convert to mesenchymal cells, an effect potentiated in a pathological environment reminiscent of fibrotic disease composed of TGF-β1 and fibronectin. Furthermore, we show that this is a Smad-dependent mechanism and implicate BMP4 and DDR1 in bronchial epithelial cell transdifferentiation.

The majority of the work on EMT in pulmonary fibrosis has so far been focused on AECs and it is less clear whether bronchial epithelial cells are able to undergo EMT. Further understanding of the EMT mechanisms in fibrosis is potentially of great importance as it could lead to approaches which impede or reverse the increase in the number of fibroblasts in fibrotic diseases.

Our work reveals that, in addition to AECII, primary HBECs are also able to convert to mesenchymal phenotypes in a Smad-dependent manner. While this work was in progress, other studies have recently also reported EMT in human airway epithelial cells. Zhang and colleagues have reported EMT-related changes in the polarized layer forming airway epithelial cell line 16HBE-14o [48] and our study extends these findings to primary HBECs. Borthwick and colleagues also support these findings [49]. In their study they demonstrated EMT of airway epithelial cells in vitro and mesenchymal cell marker expression in the epthelial cells in biopsies from obliterative bronchiolitis patients using immunohistochemistry. Furthermore, in a very recent study, airway epithelial cells isolated from asthmatics have been shown to be sensitized to TGF-β induced EMT [50]. These studies and our present work together provide supportive evidence for the occurence of EMT in airway epithelium in addition to the more extensively described process in the alveoli. Interestingly, it has been very recently reported that mouse tracheal epithelial cells can be induced to undergo EMT with TGF-β1 in vitro [51]. Whether this is the case with human tracheal cells remains to be determined. Nevertheless, a number of specialized airway and lung epithelial cell types appear to maintain an intrinsic plasticity that allows them to transdifferentiate into mesenchymal cells in pathological settings.

Our results also demonstrate that fibronectin alone is able to promote EMT in A549 epithelial cells and therefore extends the findings of Kim et al. [12] to the human setting, confirming an important role for fibronectin in the induction of EMT in human AECII. While fibronectin alone shows no significant effect in HBECs, EMT in these cells is potentiated in a pathological environment where fibronectin and excess TGF-β1 co-exist, further strengthening the relevance of our findings in disease.

Our study also gives new insight into some of the mechanism of EMT in bronchial epithelial cells. We show that DDR1 is downregulated during EMT in bronchial epithelium, whereas DDR2 is unaffected. Given that DDR1 is mostly expressed by epithelial, and not mesenchymal tissue, the reduction of its expression during EMT correlates with a decrease in epithelial identity. Remarkably, in a study comparing rat skin fetal fibroblasts of scar-free or scar-forming gestational ages, DDR1 was found to be downregulated at stages where scarring was no longer possible and collagen I was produced in excess. DDR2 levels were similar throughout gestation [52]. An analogy can be drawn with fibrosis in the adult where collagen I is overproduced and fibroblast numbers increase partly due to EMT and scarring of the tissue occurs. It therefore seems that, in situations of excess collagen and fibrosis, DDR1 can be downregulated in epithelial cells. Our findings also support recent reports on SLUG-induced EMT in Madin-Darby canine kidney cells with reduced DDR1 expression when induced to undergo EMT [53]. Shintani and colleagues (2008) [54] showed that collagen I-mediated upregulation of N-cadherin required cooperative signals from DDR1 and integrin β1 in pancreatic cells. Since we and others have shown that N-cadherin is upregulated in bronchial EMT, it will be interesting to evaluate in future studies whether integrins also play a role and whether there is any interplay with DDR1 in EMT in HBECs.

We have also investigated the effect of BMP4 during EMT of primary HBECs. Our data suggest a possible dual and context-dependent role for this growth factor. While it induced morphological EMT-like changes in HBECs, and was also able to upregulate collagen I expression, our data demonstrate that when applied in combination, BMP4 also inhibited TGF-β1-induced epithelial cell differentiation as demonstrated by its effect on MMP expression. Two epithelial cell lines were previously shown to have opposite EMT responses to BMP mediators. BEAS-2B, an airway epithelial cell line, was induced to undergo EMT when exposed to recombinant BMP4 [26, 47]. On the other hand, the A549 AEC cell line overexpressing the BMP inhibitor Gremlin also showed enhanced EMT [26, 47]. Furthermore, whereas Gremlin is over-expressed in the lungs of IPF patients and the responsiveness of IPF fibroblasts to BMP4 in culture is impaired, BMP4 and BMP7 activity is not reduced in IPF biopsies or in asbestos-exposed mouse lungs, as would be predicted by the increased expression of the BMP inhibitor Gremlin in IPF [47, 55]. The BMP4 induced mechanisms thus appear to be complex as BMP4 activity can be modulated depending on the cell type and the cytokine millieu. It is interesting to speculate that the downregulation of MMPs, after BMP treatment, may not be a direct EMT effect but a paralell mechanism caused by the inhibitory effect of BMP signalling upon proteasome function [56]. In previous studies both MMP-2 and MMP-9 have been found to be downregulated by proteasome inhibition and the proteasome pathway has previously been linked to BMP signalling [56, 57]. However, further studies are needed in order to unravel the complexities of BMP-TGFβ signalling interactions in EMT in bronchial epithelium.

The induction of the expression of MMPs, including MMP-2 and MMP-9 is well demonstrated in the context of cancer metastasis and is purported to play a role in both angiogenesis and cellular invasiveness [58] by contributing to the enhanced migratory phenotype of the cancer cell. Similarly, it was shown that, in in vitro injury models of repairing HBECs, MMP-9 accumulated in migrating HBECs at the leading edge of the wound [59]. Given the dramatic increase of MMP-9 expression that we have observed in HBECs undergoing EMT, it is reasonable to speculate that HBECs also gain a migratory phenotype during fibrosis pathogenesis in the lung. Our planned investigations will address this intriguing possibility in more detail.

Important recent advances demonstrate that several lung epithelial cell types can undergo EMT in vitro and suggest that the mechanism may also play a role in vivo during the development of various fibroproliferative diseases. Recent data from two groups using different detection methods demonstrated mesenchymal marker expression in airway epithelial cells of lung transplant patients with bronchiolitis obliterans [48, 60]. Nevertheless, it is yet to be shown conclusively that EMT of bronchial epithelial cells plays a significant role in airway tissue remodelling and peri-bronchial fibrosis in the pathogenesis of lung diseases.

Our results provide an additional insight into EMT, a potentially very important mechanism in fibrogenesis. We demonstrate that TGF-β1 can drive EMT in primary HBECs in vitro. We also show that this effect is primarily mediated via a Smad 2/3 dependent mechanism and is potentiated by cell-fibronectin interaction, in the presence of TNF-α, and can be further modulated by BMP pathway activation with BMP4. The results provided in this study establish the basis of future investigations into the mechanism of bronchial epithelial cell to mesenchymal cell differentiation, which, in turn, can contribute to a better understanding of lung fibrosis and to the development of new therapeutic approaches.