Events Involved in Epithelial Repair and EMT
In principle, epithelial repair follows different types of injury. In the case of CF, the damage to the airway epithelium can be caused by respiratory infections, in particular
Pseudomonas aeruginosa ()
[38] and inflammatory mediators
[39]. Also bacterial products secreted by
P. aeruginosa, triggered by the quorum sensing (QS) network and involved in bacterial virulence, pathogenicity, and biofilm formation, have been shown to reduce airway epithelial repair rates
[40][41][42]. On the other hand, heightened production of inflammatory mediators and reductions in anti-inflammatory molecule secretion by inflammatory and epithelial cells in CF lungs play a role in injury and remodeling of respiratory epithelia
[43][44][45].
Figure 1. Airway epithelial damage in the airways following infection with P. aeruginosa. (a) Hematoxylin and eosin-stained lung section from control mice and (b,c) hematoxylin and eosin-stained lung sections from P. aeruginosa infected mice 48 h post-intratracheal instillation at a dose of 1 × 105 colony forming units. Panel c is an enlargement of panel b. Original magnification ×20 (a,b) and ×40 (c). White arrow in (c) indicates loss of the airway epithelium.
In the airways, epithelial repair follows general rules of reconstitution whose hallmarks are
[46]: spreading and migration of neighboring epithelial cells using filopodial and lamellipodial extensions, a further step characterized by migration and proliferation of progenitor cells and, finally, differentiation. Recently, EMT induction, during which epithelial cells transform into mesenchymal-like cells, has been recognized as essential in physiologic and pathologic repair
[47][48]. During EMT, epithelial cells lose their epithelial markers (i.e., E-cadherin) and present a migratory phenotype to re-epithelialize the wound. Fibroblast growth factor (FGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), and transforming growth factor (TGF)-β are growth factors orchestrating wound repair steps and are also involved in the initiation and regulation of the EMT
[47].
The epithelium maintains its integrity through cell surface protein complexes, which contribute to form epithelial cell–cell junctions. The reorganization of the cytoskeletal architecture and polarity complexes, which result in cell shape changes, cell elongation, membrane protrusions and front–rear polarity, are essential in EMT and enable directional migration. Upon the initiation of EMT, junctions composed in the AJC are deconstructed and the junction proteins are relocalized and/or degraded
[49]. A decreased claudin and occludin expression, and the diffusion of ZO-1 from cell–cell contacts has been described in early stages of EMT
[50]. The findings of the nuclear localization of occludin and ZO-1 in CFBE cells
[16] can be now viewed in the context of partial EMT state of CF airway epithelial cells (see below). E-cadherin—one of the main components of AJs—once cleaved and detached from the plasma membrane can be degraded
[51]. Therefore, β-catenin is released from E-cadherin and act as transcription factor in the presence of WNT signaling which protects it from degradation
[52]. The decrease in E-cadherin levels can even cause the accumulation of p120 catenin (also known as catenin δ1) in the nucleus where it works as transcription factor
[53]. EMT initiation is also associated with disruption of desmosomes
[50][51], and the decrease of Cx levels which in turn induce the loss of GJs
[54]. During EMT progression, a decrease in the junction proteins expression at transcriptional level is observed, and this further stabilizes the loss of epithelial junctions
[55][56]. Moreover, the apical–basal polarity, which is mediated by molecular complexes physically and functionally integrated with the cell junction architecture, is lost.
Another key role of EMT programme is to provide cells with the ability to migrate by invading through ECM. This process requires the reorganization of cytoskeleton so as to permit the dynamic elongation and directional motility
[51][57][58]. Cells produce several types of membrane protrusion, which are rich of actin and facilitate cell movement and act as sensory extensions of the cytoskeleton. These projections include lamellipodia, which are sheet-like membrane protrusions, and spike-like extensions called filopodia at the edge of them
[59]. Invadopodia degrade ECM by proteolytic activities, thus facilitating cell invasion
[59][60]. The family of RHO GTPases is involved in these processes with RHOA promoting actin stress fibre formation and RAC1 and CDC42 promoting the formation of filopodia and lamellipodia. The conversion from apical-basal polarity (epithelial cells) to front-rear polarity (mesenchymal cells), which is one of the main aspects of EMT, involves RHO GTPases
[61][62].
The repression of genes which are fundamental for the epithelial structure (e.g., E-cadherin) is counterbalanced by the activation of genes encoding proteins involved in mesenchymal adhesion such as N-cadherin (mesenchymal neural cadherin). Depending on the cell type and the extent to which cells advance through an EMT programme, cells undergoing an EMT may begin to express vimentin, to suppress cytokeratin, to shift expression of key integrins and so forth. These changes in the intermediate filament composition enable cell motility as vimentin can interact with motor proteins
[63].
Remodelling of the ECM and changes to cell interactions with the ECM are essential in the initiation and progression of EMT. While acquiring a mesenchymal phenotype, epithelial cells lose their interaction with basal membrane and communicate with an inflammatory ECM.
As integrin complexes enable cells to receive ECM signals and integrate those elicited by growth factors, some epithelial integrins are down-regulated while others are activated in EMT. Some of these newly expressed integrins have key roles in EMT progression
[51]. Thus, α
6β
4, that mediates contacts with the basement membrane, is epigenetically down-regulated
[64], whereas β
1 integrins increase during EMT
[65][66][67][68]. During wound repair, β1-integrin subunit was found to be expressed by repairing cells on their basolateral side as well as on the apical side
[69]. Kim and colleagues
[65] demonstrated the crucial role of the laminin receptor α
3β
1 integrin in E-cadherin turnover and the remarkable crosstalk and interdependence of TGF-β signalling and β-catenin–SMAD signalling systems, in the control of EMT. Also the fibronectin receptor α
5β
1 integrin expression is augmented during EMT increasing cell adhesion to fibronectin, the expression of which is also activated during EMT, and promoting cell migration and protection from apoptosis. The increased expression of the β
1 integrins and their ligation of collagen type I initiates a signaling cascade, leading to disassembly of the E-cadherin adhesion complex and the nuclear translocation of β-catenin, ultimately determining cell proliferation
[68]. A strict connection of changes to the integrin repertoire and ligation as well as intracellular signals and increased expression of metalloproteinases (MMP2 and MMP9) has been demonstrated
[49][70][71]. MMPs will enhance ECM protein degradation where integrin adhesion receptors focally interact with inflammatory ECM proteins, thus enabling invasion
[51][59][72]. Other effects of MMPs related to the EMT activation are related to the trimmering of the extracellular domain of E-cadherin, thus contributing to the loss of AJs
[70], and to increased SNAIL1 expression operated by increased levels of reactive oxygen species
[73]. Finally, it has to be recognized that some growth factors are stored in the ECM and that localized ECM degradation may release them, such as is the case for TGF-β that is present in a latent form and is activated by MMPs and α
vβ
6 [74][75]. TGF-β, in turn, stimulates the expression of collagens and fibronectin, which are involved in the matrix remodeling.
EMT is induced by an interplay of soluble growth factors such as HGF, members of the TGF (e.g., TGF-β) and FGF families, insulin-like growth factor (IGFs, e.g., IGF-1), EGF as well as extracellular matrix such as collagen or hypoxic conditions. These factors activate signaling pathways leading to either expression or post-transcriptional and post-translational modification of EMT-associated transcription factors (EMT-TFs)
[76][77]. Three main families of EMT-TFs have been described with the SNAI (SNAI1/Snail and SNAI2/Slug), ZEB (ZEB1 and ZEB2), and TWIST (TWIST1 and TWIST2) nuclear proteins, playing pivotal roles in the orchestration of EMT
[78]. These EMT-TFs have been shown to interact with a variety of proteins involved in transcriptional regulation including proteins that function in epigenetic modification, forming together regulatory complexes.
In the airways, the final step of wound repair is the terminal differentiation which means the reconstitution of a pseudostratified epithelium with mucus-secreting cells (goblet), secretory (Club cells), ciliated cells, PNEC (pulmonary neuroendocrine cells), and basal cells. Recent studies have identified another cell type which has been called “ionocyte” and expresses higher levels of CFTR than other airway cells do
[33][34]. Ionocytes derive directly from basal cells (BCs), some of which appear to function as classic multipotent stem cells, while other BCs are thought to be progenitors already committed to a ciliated or secretory fate
[79][80]. As genetic (single-cell RNA sequencing) and more detailed histological and functional studies advance, not only ionocytes but also other rare cell types derived from BCs (PNEC, tuft cells, mucous ciliated cells and deuterosomal cells) are being studied during airway epithelium regeneration and repair
[81][82].
The processes of epithelial repair are modulated by several growth factors, including EGF and related cytokines (TGF-α, amphiregulin, heparin-binding EGF), which through EGF receptors (EGFR), produce motility, proliferative responses, differentiation and survival
[43][83][84]. MMPs and a disintegrin and metalloproteases (ADAMs) release from the cell surface EGFR ligands, including TGF-α, heparin-binding EGF (HB-EGF) and amphiregulin (AREG), that in turn can bind and activate EGFR in an autocrine or paracrine manner, while the transmembrane (pro) form may activate EGFR in adjacent cells (juxtacrine)
[85]. During migration, cells attach to a provisional matrix of fibronectin and other extracellular matrix (ECM) components such as fibrin and fibrinogen. Platelet-derived growth factor (PDGF) and TGF-β act as the main modulators of ECM deposition by attracting and activating fibroblasts, in this way regulating airway repair
[86][87]. Migrating epithelial cells overexpress MMPs to degrade the provisional matrix which provides novel attachment sites for lamellipodia. MMP-9 (gelatinase B), MMP-3 and MMP-11 (stromelysin 1 and 3, respectively), and MMP-7 (matrilysin), have been implicated in the matrix remodeling and acquisition of a typical epithelial-mesenchymal phenotype
[88]. It has been suggested that the high expression and activation levels of MMPs can be regulated by an increase in IL-8, a pro-inflammatory cytokine
[89]. TGF-β plays also a role in this remodelling process by up-regulating MMP-2
[90]. Furthermore, epithelial repair is stimulated by other growth factors, namely insulin, IGFs, HGF, keratinocyte growth factor (KGF), calcitonin gene-related peptides, and the cathelicidin LL-37 peptide. HGF and KGF acts as chemotactic and growth-stimulating factors. They also stimulate the synthesis of ECM and facilitate interactions with MMPs through specific cell receptors
[91][92]. The main features of regeneration and wound repair in a non-CF airway epithelium are represented in .
Figure 2. Wound repair and regeneration in a non-CF airway epithelium. Following injury, many cytokines and growth factors (e.g., EGF, HGF, FGF) are secreted in the wound repair microenvironment, which incite in basal cells a migratory and proliferative phenotype. Shedding of EGFR ligands (e.g., EGF) by ADAMs and binding to EGFR in an autocrine or iuxtacrine manner are key events involved in stimulation of cell migration and proliferation. Afterwards, MMP secretion by regenerating epithelial cells and subsequent TGF-β activation lead to genetic expression changes, related to EMT stimulation and activation of EMT-transcription factors (EMT-TFs). Epithelial cells and macrophages release TGF-β that induce ECM component deposition by epithelial cells and stimulate fibroblast activation, resulting in further matrix deposition. These events provoke alterations in junctional complexes and reorganization of actin cytoskeleton (not shown), modification of various integrin expression with β1-integrins increase at basal side and ectopic expression on the apical side, deposition of inflammatory ECM glycoproteins (e.g., fibronectin is shown) and its remodeling exerted by MMPs. TGF-β and MMPs enhance each other in a positive way. This process proceeds with the formation of a squamous stratified epithelium and subsequent pseudostratification and mucociliary differentiation.