The EMT process has been described in proliferative vitreoretinopathy (PVR) and wet age-related macular degeneration (AMD) [55]. PVR is the most common cause of failed retinal detachment repair and is characterised by a sequence of inflammatory and fibrotic mechanisms [56]. Retinal pigment epithelium (RPE) cells, a cellular monolayer composed of mitotically quiescent cells, are known to de-differentiate and lose their fully matured state as a result of a variety of stresses, including oxidative stress and mechanical dissociation of cell–cell junctions [57]. Dissociation of cultured RPE cells leads to morphological changes of the cells into fibroblast-like cells through the activation of the EMT programme [57]. During this process, the RPE cells, trans-differentiated into mesenchymal cells, show increased motility and enhanced ability to proliferate and acquire resistance to apoptosis and the capacity to produce extracellular matrix proteins [57]. RPE cells undergoing EMT contribute to scarring and wound contractions in PVR as well as subretinal fibrosis in advanced AMD [58], which is characterised by the formation of choroidal neovascularisation (CNV) [59]. Several ADAMs seem to be involved in these processes whose expression is variable within the retina; both ADAM10 and ADAM17 are widely expressed during the embryonic period in the different layers of the retina, whereas ADAM12 is mainly expressed in the ganglion cell layer in a later stage of development [60]. The ADAM10, in particular, is activated by TGF-β1 and, specifically, determines the E-cadherin cleavage [61]. The shedding of E-cadherin modulates the activation of the β-catenin signalling pathway, which is involved in the pathogenesis of several fibrotic diseases [62]. Furthermore, both ADAM10 and ADAM17 are involved in the regulation of the Notch-mediated signalling EMT programme important during retina development [63].

Important investigations in this field were conducted by Park et al. on Epstein Barr-transformed adult RPE cells that showed a spindle-like shape phenotype that expresses several mesenchymal markers and secretes TGF-β and VEGF [55]. These cells lose expression of E-cadherin and N-cadherin, which is the most common cell–cell junction proteins in RPE cells [64], gaining expression of mesenchymal markers, such as vimentin and/or a-SMA. Using these transformed cells, the authors investigated the molecular mechanisms of EMT in PVR or CNV conditions using the multi-kinase inhibitor Sorafenib (SRF, Nexavar; Bayer HealthCare Pharmaceuticals, Inc., Whippany, NJ, USA) to study the effects on the regulation of EMT by ADAM proteins. SRF, initially approved for the treatment of advanced renal cell carcinoma and hepatocellular carcinoma (HCC), was recently demonstrated to have antifibrotic activity in vitro [65,66,67].

SRF inhibits STAT3 phosphorylation in a variety of tumours, including HCC [68,69,70]. Moreover, SRF also inhibits TGF-β-induced STAT3 activation during TGF-β-mediated EMT in mouse hepatocytes [67]. Data collected are promising because they show that, in transformed RPE cells, SRF is able to downregulate migration-related signalling molecules, such as HIF-1a, p-STAT3 and MMP2; this process seems to be nardilysin (NRD-1)-dependent. NRD-1, a zinc peptidase of the M16 family localised diffusely in the cytoplasm and secreted to the cell surface [71], binds to the extracellular domain of ADAM17, determining its catalytic activity [72]. This role of NRD-1 was confirmed by NRD-1 knockdown that downregulates the EMT process in EBV-transfected RPE cells [55] (the mechanisms hypothesised are reported in Figure 3). Obviously, the discovery of the precise mechanisms that govern the acquisition of the EMT phenotype from RPE cells in retinal diseases would likely be useful to identify new therapeutic approaches.

Recent experimental evidence reports that ADAM17 is involved in chronic kidney disease (CKD), playing a pro-inflammatory and pro-fibrotic role [73]; blocking ADAM17 activity is, in fact, fibrosis and inflammation resulted attenuated, suggesting ADAM17 as a possible new valuable therapeutic target in CKD treatment. In addition, ADAM17 expression is also variable within the renal parenchyma and seems to be highly expressed in distal renal tubules and increased in the whole kidney in diabetic experimental mice [74].

Diabetic nephropathy is a major cause of chronic kidney disease and kidney failure. Although the kidney undergoes pathological changes in all its compartments, the earliest manifestation of glomerular sclerosis is the deposition of the ECM protein [75]. High glucose (HG) is the base of diabetic nephropathy by determining ECM production in glomerular mesangial cells. TGFβ1 is a major mediator of the HG-induced fibrotic response, and renal cells cultured in HG condition determines phosphorylation and the nuclear translocation of Smad3 [76]. In this scenario, ADAM17 mediates the HG-induced TGF-β1 upregulation and ECM protein production in kidney cells acting on the release of ligands for the EGFR [77,78]. ADAM17 activation is, in fact, required for HG-induced upregulation of the pro-fibrotic cytokine TGF-β1 [78,79]. Researchers demonstrated that the phosphorylation in two C-terminus sites of ADAM17 seems to be involved in the activation of the pro-fibrotic responses of ADAM17. HG also induced furin-dependent maturation of ADAM17 on the cell surface [79]. Furthermore, HG-induced ADAM17 activation requires the upstream regulator focal adhesion kinase (FAK) [79]. FAK acts, recruiting both Src and PI3K, with the subsequent phosphorylation of ADAM17. These studies suggest that the inhibition of ADAM17 activation through targeting HG-specific activators such as FAK or acting on FAK interaction with ADAM17 could represent innovative fields of investigation for the treatment of diabetic nephropathy (Figure 4).

The process of cancer-associated EMT, consisting of the loss of cell–cell junctions, decreases in the epithelial markers, increases expression of mesenchymal markers and cytoskeleton rearrangement, which leads to an increase in cellular invasiveness. Additionally, these changes go hand in hand with the secretion of MMP-2/-9 and FAK [80]. MMP-2/-9 are proteolytic enzymes that degrade the ECM proteins, thus modifying ECM composition and acting directly on cell surface molecules, determining EMT activation [81]. ADAM17 overexpression elevates the expression of MMP-2 and MMP-9, while ADAM17 knockdown downregulates the expression of the same MMPs. This evidence clearly suggests that ADAM17, through the elevation of the MMP-2 and MMP-9 expression, accelerates EMT [7]. ADAM17 knockdown resulted, furthermore, in the downregulation of vimentin, Snail and N-cadherin and the upregulation of E-cadherin; in contrast, ADAM17 overexpression led to the upregulation of vimentin, Snail, N-cadherin and downregulation of E-cadherin, confirming that ADAM17 promotes EMT in gastric carcinoma cells [7]. As TGF-β/Smad signalling is closely related to EMT in cancer [82], recent investigations reported that ADAM17 knockdown downregulated TGF-β and p-Smad2/3 in gastric carcinoma, while ADAM17 overexpression resulted in the upregulation of TGF-β and p-Smad2/3, but without having any effect on total Smad2/3 protein [7]. Hence, this is a confirmation that ADAM17 promotes EMT probably via TGF-β/Smad signalling in gastric carcinoma (Figure 4).

In conclusion, ADAM17 promotes proliferation, migration and invasion in gastric carcinoma cells. Importantly, the results detail a mechanism (reported in Figure 4) of ADAM17-mediated EMT through upregulating TGF-β/Smad signalling pathway. These findings suggest that ADAM17 might be an important therapeutic target candidate in gastric cancer.

Liver fibrosis is characterised by an excessive accumulation of ECM or scar tissue. The liver resident mesenchymal cells, particularly hepatic stellate cells (HSCs), have been described to be the primary source of ECM in liver fibrosis. As a consequence of liver damage, HSCs start to proliferate and undergo their differentiation into myofibroblasts that have a proliferative, contractile and fibrogenic phenotype [83]. Activated HSCs are characterised by the expression of various specific molecules such as α-SMA, desmin, glial fibrillary acidic protein, platelet-derived growth factor receptor and a massive amount of collagen I [84]. Activated HSCs, once transformed into myofibroblast-like cells, promote chronic inflammation, leading to cirrhosis and HCC. HSCs are major cellular components of HCC stroma, where they modulate the proliferation and invasiveness of cancer cells and are considered the primary source of EMT-dependent fibrogenic myofibroblasts in the injured liver [85,86]. This switch in HSCs differentiation was mediated by ADAM proteases, and different molecular mechanisms have been reported related to ADAM-mediated EMT in the liver [87,88]. ADAM17 performs its role in promoting EMT of HCC through the activation of the Notch signalling pathway, which occurs after Notch proteolytic cleavage and active Notch intracellular domain (NICD) release [89]. Recent discoveries report that the pro-EMT effects of ADAM17 were antagonised by specific micro-RNA whose anti-EMT effects determined a decreased expression of mesenchymal markers (N-cadherin and Vimentin) and of pro-EMT transcription factors (ZEB1, SNAIL and TWIST), with a concomitant increased expression of epithelial marker E-cadherin [90]. Based on this evidence, researchers investigated the use of ADAM17 inhibitors, such as ZLDI-8, showing their ability to prevent EMT in HCC cells by decreasing the release of Notch NICD, thereby improving the therapeutic efficacy of anticancer drugs [90,91,92]. Therefore, ADAM17, involved in the transactivation of Notch signalling in liver cancer stem cells, seems to contribute to the enhancement of HSCs aggressiveness [93]. In addition, Notch signalling was implicated in the ADAM17-dependent activation of integrin β1, thereby promoting the proliferation, migration and invasion of HCC cells [94]. In the context of these new findings, a new G-protein-coupled receptor 50 (GPR50)-mediated regulation of ADAM17-induced Notch signalling in HCC progression was also demonstrated [95]. Data collected so far suggest that ADAMs have been implicated at all stages of HCC progression, starting from inflammation and subsequently through to fibrosis, angiogenesis, proliferation, EMT and invasion (Figure 4). The spatial and temporal dynamics of ADAMs activation and their mechanisms of action are, however, still insufficiently characterised, and future work is needed, focusing on better characterising the ADAMs’ contribution to HSCs differentiation in the HCC progression.

Idiopathic pulmonary fibrosis (IPF) is a type of interstitial lung disease that is prevalent in elder smokers. The phases of IPF include alveolar epithelial cell damage and activation, inflammatory cell infiltration, EMT initiation and ECM protein accumulation [96]. During the progression of IPF, most fibroblasts originate from lung epithelial cells, which undergo EMT and play a crucial role in fibrotic disease progression. The TGF-β signalling pathway has been suggested to contribute to the EMT process and produce ECM proteins, such as fibronectin (FN) [97]. Therefore, TGF-β and EMT may be a hallmark of fibroblast activation.

As reported above, ADAM17 is responsible for the cleavage of extracellular domains of substrate proteins [54], thus regulating some important physiological and pathophysiological processes and the expression of membrane-bound proteins such as cytokines and growth factors [98]. Increased ADAM17 expression is identified in several inflammatory diseases, cancers, and organ fibrotic changes, including IPF [35,36,99,100]. During the progression of chronic IPF, the volume and ventilation of the lungs are gradually decreased due to abnormal proliferation of fibroblasts through the EMT process, which causes collagen deposition and finally leads to architectural distortion [101]. A recent study demonstrated that ADAM17 regulates TGF-β-mediated EMT through the cleavage of vasorin (VSN), a type I transmembrane protein initially identified in screening to isolate novel proteins containing a signal sequence. VSN was shown to attenuate TGF-β signalling by sequestering the growth factor, and its expression was found restricted to the aorta, kidney and placenta [102,103]. In addition, ADAM17 is responsible for the angiotensin-converting enzyme 2 (ACE-2) ectodomain shedding occurring in lung fibrogenesis, demonstrating that ADAM17 certainly participated in IPF [103]. However, the role of ADAM17 in TGF-β-induced EMT in IPF remains uncertain.

Recently, new impetus has been given to research in this field, finding that connective tissue growth factor (CTGF), an immediate-early protein mediated by TGF-β, regulates the growth of fibroblasts and the secretion of ECM [104]. A previous study suggested that subcutaneous co-injection of TGF-β plus CTGF induced sustained fibrosis in mice [105]. In lung tissue obtained from IPF patients, an enhanced expression of both CTGF protein and mRNA was observed [106], and, moreover, CTGF/integrin-linked kinase signalling mediates the activation of EMT in lung alveolar epithelial cells [107]. Data recently collected revealed an unexpected role for ADAM17 in the regulation of this phenomenon, showing that TGF-β might activate ERK, ADAM17 and Ribosomal S6 kinase-1 (RSK1) signalling pathways; this activation cascade determines the phosphorylation of the enhancer-binding protein β (C/EBPβ) that binds the CTGF promoter region, leading to CTGF synthesis and expression. These investigations start from several lines of evidence of the role of RSK1 and protein kinase C (PKC) in the phosphorylation of C/EBPβ, a transcription factor that participates in the modulation of pro-inflammatory protein expression [108,109]. Moreover, CTGF participates in the mechanism leading to TGF-induced FN expression in human lung epithelial cells [110]; in an experimental model represented by TGF-β-induced renal fibrosis in mice, this mechanism was inhibited, blocking MEK activity and so attenuating CTGF expression [111]; however, it remains unclear as to whether RSK1 and C/EBPβ are involved in TGF-β-induced CTGF expression in human lung epithelial cells and what is ADAM17’s role in EMT activation in the lung. Starting from the results obtained from Blom et al. [104], suggesting that CTGF acts as a modulator of TGF-β-dependent fibrogenesis and EMT activation in lung epithelial cells [104], studies have since progressed; it was demonstrated that TGF-β-induced CTGF expression in human lung epithelial cells provides the participation of ERK, ADAM17, RSK1 and C/EBPβ [110], and both ADAM17 and CTGF seem to mediate TGF-β-induced FN expression [110]. It is clear now the involvement of ADAM17 in the TGF-β-induced expression of CTGF and EMT in the lung; in fact, ADAM17 gene silencing reduced TGF-β-induced CTGF and FN expression in human alveolar basal epithelial cell line [110]. Furthermore, through the use of CTGF gene knockdown, a reduced TGF-β-induced FN expression was observed, confirming the correlated importance of ADAM17 and CTGF in TGF-β-induced FN expression in human lung epithelial cells [110]. Further clarifications on the molecular mechanisms involved in IPF fibrosis emerged through studying the ERK pathway activation that seems to regulate the expression of pro-fibrotic proteins in IPF such as osteopontin [112]. By using a specific ERK inhibitor, TGF-β-induced CTGF expression was reduced in the alveolar basal epithelial cells to levels comparable to those obtained through the use of RSK1 gene silencing; additionally, TGF-β enhanced the phosphorylation of ERK and RSK1 that was decreased by using ERK inhibitors [110]. This experimental strategy allowed for the demonstration that ERK mediates TGF-β-induced ADAM17 phosphorylation, and ADAM17 regulates TGF-β-induced RSK1 phosphorylation; overall, these results suggest that the ERK/ADAM17/RSK1 signalling pathway activation was required for TGF-β enhanced CTGF expression in the lung [110].

The same ERK/ADAM17/RSK1 pathway has a positive effect on C/EBPβ phosphorylation and activation, and ADAM17 plays a key role in TGF-β-induced CTGF expression and EMT through the ERK/RSK1/C/EBPβ pathway [110].

In conclusion, data collected evidenced that TGF-β activates the ERK/ADAM17/RSK1/C/EBPβ signalling pathway, after which it promotes the link of C/EBPβ to the C/EBPβ site on the CTGF promoter region to regulate CTGF expression in human lung epithelial cells, revealing a signalling pathway related to ADAM17-dependent EMT and fibrosis, which may provide a new therapeutic orientation for the treatment of IPF (Figure 5).