This is the first study to demonstrate that components of the Hippo signaling pathway are required for E-cadherin-mediated contact inhibition of proliferation in the lungs after air pollution exposure. Air pollution caused E-cadherin deletion by HMGB1; this suppressed Yap activation which triggered senescence, alveolar epithelial differentiation, and apoptosis. These protein interactions were also supported by our proteomics approach. These results provide evidence that E-cadherin hemophilic binding regulates the Hippo pathway in the lungs after air pollution exposure. Furthermore, these protein expressions in the lungs due to air pollution were consistent with emphysematous lungs of COPD patients. The underlying pathways regulated by air pollution could be vital in the development of emphysema.
The lungs are the first target organ to interact with air pollution after inhalation. Impairment of properties of the epithelial barrier is the main hallmark in the development of lung diseases, such as COPD and asthma. HMGB1 is a nuclear DNA-binding protein, which is secreted into the extracellular milieu and functions as a proinflammatory cytokine [20]. Recent reports showed that HMGB1 is involved in inflammatory lung diseases [21, 22]. Increasing permeability in the lungs due to air pollution exposure has been widely observed [23, 24]. Our study observed that air pollution increased levels of HMGB1 in the lungs, which disrupted properties of the epithelial barrier due to deletion of E-cadherin. The association between HMGB1 and E-cadherin was further confirmed by E-cadherin-knockdown and HMGB1 treatment in A549 cells. A previous study showed that HMGB1 damaged the airway epithelial barrier due to E-cadherin deletion [25], and the impairment was further aggravated by interleukin (IL)-1β. Collectively, air pollution exposure causes an interplay between HMGB1 and E-cadherin in the lungs. This could result in disruption of epithelial barrier properties in the lungs.
Previous reports showed that E-cadherin mediates contact inhibition of proliferation via the Hippo signaling pathway [17, 18]. Yap and Taz, Hippo signaling pathway components, are transcriptional coactivators that are the main downstream mediators of the Hippo pathway [26]. In this study, we further showed that exposure to air pollution reduced E-cadherin expression and Yap phosphorylation in the lungs. Also, both exposure to DEPs and E-cadherin-knockdown in A549 cells reduced Yap phosphorylation. Depletion of E-cadherin-bound β-catenin decreased Yap phosphorylation (S127 residue) and Yap nuclear accumulation [17]. Furthermore, E-cadherin homophilic binding occurs independent of other cell interactions, and this is able to control the subcellular localization of Yap [27]. Together, our results suggest that E-cadherin is an upstream cell-surface receptor that regulates Hippo signaling pathway components, which may control alveolar epithelial differentiation and apoptosis after air pollution exposure.
Alveolar epithelial differentiation from type II to type I is not yet clear; this is how E-cadherin homophilic ligation stimulates the Hippo pathway after air pollution exposure. Type II alveolar epithelial cells are considered to be progenitor cells involved in regenerative processes [28] via the Hippo signaling pathway [13, 14]. When the Hippo pathway is inhibited, Yap/Taz are able to accumulate in nuclei. The accumulated Yap/Taz interact with transcription factors and activate gene expressions, thereby activating cell survival, proliferation, and differentiation pathways [29]. A previous report showed that inactivation of E-cadherin and α-catenin affected bronchiolar progenitor cell differentiation and airway regeneration [38]. Indeed, we observed significant reductions in SPC and T1α in the lungs after air pollution exposure. Our results suggest that the pathway for type II-to-type I alveolar epithelial differentiation is impaired by chronic exposure to air pollution. Also, numbers of these epithelial cells were decreased by air pollution. Thus, air pollution is involved in regulating the Hippo signaling pathway which affects AECII activities and could lead to alterations in its proliferation.
Cellular senescence is a state of permanent inhibition of cell proliferation, which is associated with COPD [30]. We observed that air pollution exposure reduced Sirt1 phosphorylation in the lungs. Also, both exposure to DEPs and E-cadherin-knockdown in A549 cells caused a decrease in Sirt1 phosphorylation. Air pollution induces senescence by regulating Sirt1 as observed in vitro [31] and in vivo [32]. Senescent cells were shown to secrete a senescence-associated secretory phenotype (SASP). HMGB1 is linked to regulation of cell senescence, which is considered a mediator of cellular senescent responses [33, 34]. Alveolar epithelial cell senescence was reported in COPD patients [35], which may explain the abnormal cell turnover that promotes the loss of alveolar cells in emphysematous lungs.
Apoptosis and senescence are cellular responses to a variety of intrinsic and extrinsic signals. In contrast to apoptosis, cellular senescence is the loss of proliferative capacity. Our results indicated that apoptosis via p53 expression occurred due to air pollution exposure, which was confirmed by DEP exposure and E-cadherin-knockdown in A549 cells. Previous reports indicated that p53 is required for PM-induced apoptosis of alveolar epithelial cells [36, 37]. Increased levels of apoptosis in alveolar epithelia lead to emphysematous changes in the lungs [38, 39]. Recent studies showed that emphysema is associated with disruption of functions of alveolar apoptosis and cell proliferation [38]. Not only is apoptosis activated by air pollution, but we also found cellular senescence by Sirt1 phosphorylation in the lungs and in A549 cells. These results suggest that air pollution disables the proliferative capacity of lung cells. Regulation of both apoptosis and senescence was associated with E-cadherin expression according to results of E-cadherin-knockdown in A549 cells. Therefore, air pollution can cause apoptosis and cellular senescence, and this could occur by E-cadherin mediating contact inhibition of proliferation via the Hippo signaling pathway.
The PPI network was examined in the lungs using proteomic analyses. We confirmed that E-cadherin plays an essential role in activating the Hippo signaling pathway and regulating cell apoptosis, senescence, and differentiation of ACEII to ACEI due to air pollution. First, we found that MMP-9 was involved in regulation between HMGB1 and E-cadherin. This finding showed that air pollution can induce the epithelial-to-mesenchymal transition (EMT) by HMGB1 expression. A previous study observed that HMGB1 induced the EMT by downregulating Cdh1 gene expression in human airway epithelial cells [40]. This was confirmed by our results, in which E-cadherin regulated apoptosis and senescence due to air pollution. Notably, cellular differentiation from AECII to AECI was regulated via Krt5 from E-cadherin. Another report identified that SPC and Krt5 were in airway and alveolar cells in lung disease, indicating that alternative progenitor lineages are mobilized to regenerate the alveolar epithelium when AECII is severely injured [41]. The Hippo signaling pathway component, Yap, was identified to regulate apoptosis and senescence via Smad3 in this study. This result suggests that the Hippo pathway is not only essential to cell death and proliferation due to air pollution, but also important for the EMT.
We observed that unconcentrated ambient air pollution exposure in rats for 6 months caused significant emphysema. This observation was also reported by our previous study [42]. We further investigated these protein expressions in alveolar regions of control and COPD subjects. Notably, we found that PM had been deposited in COPD lungs, but not in control subjects. We suspect that inhaled PM is more easily deposited or trapped in the lungs due to a reduction in lung functions due to COPD severity. One study showed that higher fractions of PM from cigarette smoke were deposited in the last few airway generations [43]. The deposited PM in the lower airway and alveolar regions may be associated with the development of emphysema. However, more evidence is required to confirm the possible causal relationship.
We observed that expressions of E-cadherin with Yap, and SPC with T1⍺ in alveolar regions of COPD subjects obviously decreased compared to those regions of control subjects. This observation is consistent with results in rats after air pollution exposure. Previous reports indicated that selective loss of Yap in restricted regions of the mouse lung epithelium leads to lung cysts that mimic emphysema [16]. Additionally, a Taz deficiency resulted in lung developmental abnormalities in mice, which led to an emphysematous lung phenotype [15]. Collectively, air pollution regulates E-cadherin which mediates contact inhibition of proliferation via the Hippo signaling pathway in emphysema.
There are some limitations of this study that should be noted. Chemical effects of air pollution on the Hippo signaling pathway were not examined in this study, and should be determined in the future. Primary AECII should be used to understand the ability of differentiation to ACEI by air pollution. The role of the air pollution-induced EMT should be further investigated. Confirmatory experiments linked to air pollution should be conducted in humans.