The present study demonstrated that CAY10603 improved emphysema and airway inflammation induced by cigarette smoke both in vivo and in vitro. The mechanism of action of HDAC6 may be associated with regulating epithelial barrier dysfunction and reversing EMT via TGF-β1/Smad2/3 signaling pathway.
As mentioned above, EMT refers to the process in which epithelial cells lose polarity and turn into mesenchymal cells in special conditions[32]. Current studies [33] suggested that airway remodeling in COPD was mainly related to type 2 EMT, involving tissue fibrosis. Similar to EMT, endothelial to mesenchymal transition (EndMT) has been reported to be involved in vascular remodeling in COPD [34]. The mechanism of EMT has been extensively studied in the pathogenesis of infiltration and metastasis of tumor cells. Current studies have found that EMT exists in small airway epithelium of COPD patients [35], and EMT may play an important role in the occurrence and development of airway remodeling. Smoking is one of the most important causes of COPD [36], and current studies mainly focus on the effect of CS on EMT in COPD. There are various pathways through which CS acts on EMT, oxidative stress, destruction of cell connections, and destruction of cytoskeleton structure may all participate in this process. Apoptosis mediated by reactive oxygen species (ROS) could act on EMT through the PI3K/AKT/NFKB/MMP-9 signaling pathway in cancer [37]. In COPD, ROS could promote epithelial phenotypic transformation, resulting in abnormal proliferation and differentiation of epithelial cells, leading to subepithelial collagen deposition [38]. Current studies have observed that CS can lead to EMT in both alveolar and airway epithelium. CS could promote EMT in alveolar epithelial cells through WNT/β-catenin signaling pathway, resulting in impaired alveolar repair ability [39]. COPD begins with small airway dysfunction, so our study focused on the change of small airway and airway epithelium in COPD. The mechanism of CS promoting EMT in airway epithelial cells is also under study, which may involve TGF-β/Samd [40], WNT/β-catenin, Hedgehog (Hh) [41], urokinase plasminogen activator receptor (uPAR) [42], Notch signaling pathways [43] and other signaling pathways. Among them, the TGF-β/Samd pathway has been relatively studied. In brief, TGF-β mainly phosphorylates the Smad complex, which can translocate into the nucleus and promote the expression of EMT transcription-induced genes [14]. In our study, similar manifestations were also found, such as increased TGF-β in BALF after CS exposure and increased content of pSmad2/3 in cell experiments.
The barrier function of the airway epithelium, a structure of interconnected cells that form the first barrier against environmental damage, maintained by tight junctions (TJs) and adherens junctions (AJs), limits the permeability of inhaled pathogens and environmental stressors [44]. TJs, the apical portion of the cell surface, are composed of transmembrane proteins claudin (CLDN), Occludin (OCLN), and junction adhesion molecules (JAMs) [45]. In addition, zonula occludens (ZO)-1, ZO-2, ZO-3, Par-3, Par-6 are also associated with TJs formation [46]. TGF-β1 has been shown to prevent CSE-induced tight junction disruption and barrier function loss. ZO-1 and ZO-2 protein levels were restored, after TGF-β1 treating to exposure of human bronchial epithelial cells (16HBE14o−) to CSE [47]. Other cell experiments [48] have shown that HDAC6 could deacetylate the promoters of tight junction genes in the nucleus, leading to the dissolution of tight junction. Our experiment also observed the decrease of ZO-1 and Occludin in IHC and Western blot in the CS group, while CAY10603 treatment showed significant improvement, indicating that CAY10603 can adjust TJs through regulating the expression of ZO-1 and Occludin.
As a member of histone deacetylases (HDACs), HDAC6 functions as a deacetylase that mainly targets non-histone proteins in the cytoplasm and non-enzymatic functions regulated by the ubiquitin proteasome system [49]. So far, various substrates of HDAC6 have been found, including α -tubulin, cortactin, Hsp90, β -catenin, RIG-I, Ku70, HSPA5, HMGN2, PrxI, Tat and so on [50]. HDAC6 is associated with the occurrence and development of a variety of diseases, including neurodegenerative diseases [51], cancer [52], cardiovascular diseases [53], renal fibrosis [54], cystogenesis [55], inflammation [56], etc. Among respiratory diseases, studies mainly focus on lung cancer, whereas there are few studies related to COPD. Wang et al.[57] found that HDAC6 made epidermal growth factor receptor (EGFR) deacetylated and played an important role in the control of cell proliferation in lung adenocarcinoma. A study by Deskin et al.[58] found that HDAC6 regulated EMT in non-small cell lung cancer (NSCLC) by mediating TGF-β-Notch signaling cascade. These signaling pathways also regulate EMT in COPD, so our experiment explored the role of HDAC6 in regulating EMT in COPD. Lam et al.[59] found that HDAC6 was an important regulator of autophagy-mediated ciliary shortening during CS exposure. A study by Su et al.[60] suggested that HDAC6 up-regulated collagen synthesis and proliferation of bronchial smooth muscle cells (BSMCs), leading to airway remodeling in COPD patients. In our experiment, we found that CAY10603 treatment could reduce the expression of E-cadherin and increase the expression of α-SMA induced by TGF-β1 in HBE cells. This effect was achieved through the suppression of Smad2 and Smad3 phosphorylation. CAY10603 attenuated TGF-β1-induced EMT in HEB cells and is expected to be a potential treatment for COPD in the future.
However, there are still some limitations to our study. For example, our study did not fully clarify how HDAC6 acts on tight junction proteins. Meanwhile, whether HDAC6 regulates EMT of airway epithelial cells through other mechanisms, such as adherens junctions, will be the focus of our subsequent research.